Reversing the formation of advanced glycosylation endproducts

ABSTRACT

The present invention relates to compositions and methods for inhibiting and reversing nonenzymatic cross-linking (protein aging). Accordingly, compositions are disclosed which comprise an agent capable of inhibiting the formation of advanced glycosylation endproducts of target proteins, and which additionally reverse pre-formed crosslinks in the advanced glycosylation endproducts by cleaving alpha-dicarbonyl-based protein crosslinks present in the advanced glycosylation endproducts. Certain agents useful are thiazolium salts. The method comprises contacting the target protein with the composition. Both industrial and therapeutic applications for the invention are envisioned, as food spoilage and animal protein aging can be treated. A novel immunoassay for detection of the reversal of the nonenzymatic crosslinking is also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a Divisional of Ser. No. 08/588,249, Jan. 18, 1996,now U.S. Pat. No. 5,853,703, which is a continuation-in-part of Ser. No.08/473,184, Jun. 7, 1995, abandoned, which is a continuation-in-part ofSer. No. 08/375,155, Jan. 18, 1995, U.S. Pat. No. 5,656,261.

BACKGROUND OF THE INVENTION

The present invention relates generally to the aging of proteinsresulting from their reaction with glucose and other reducing sugars,and more particularly to the inhibition of the reaction ofnonenzymatically glycosylated proteins and the breaking of cross-linkedformed subsequent to formation of advanced glycosylation (glycation)endproducts.

The reaction between glucose and proteins has been known for some time.Its earliest manifestation was in the appearance of brown pigmentsduring the cooking of food, which was identified by Maillard in 1912,who observed that glucose or other reducing sugars react with aminoacids to form adducts that undergo a series of dehydrations andrearrangements to form stable brown pigments. Further studies havesuggested that stored and heat treated foods undergo nonenzymaticbrowning as a result of the reaction between glucose and the polypeptidechain, and that the proteins are resultingly cross-linked andcorrespondingly exhibit decreased bioavailability.

This reaction between reducing sugars and food proteins was found tohave its parallel in vivo. Thus, the nonenzymatic reaction betweenglucose and the free amino groups on proteins to form a stable,1-deoxyketosyl adduct, known as the Amadori product, has been shown tooccur with hemoglobin, wherein a rearrangement of the amino terminal ofthe beta-chain of hemoglobin by reaction with glucose, forms the adductknown as hemoglobin A1c. The reaction has also been found to occur witha variety of other body proteins, such as lens crystallins, collagen andnerve proteins. See Bucala et al., "Advanced Glycosylation; Chemistry,Biology, and Implications for Diabetes and Aging" in Advances inPharmacology, Vol. 23, pp. 1-34, Academic Press (1992).

Moreover, brown pigments with spectral and fluorescent propertiessimilar to those of late-stage Maillard products have also been observedin vivo in association with several long-lived proteins, such as lensproteins and collagen from aged individuals. An age-related linearincrease in pigment was observed in human dura collagen between the agesof 20 to 90 years. Interestingly, the aging of collagen can be mimickedin vitro by the cross-linking induced by glucose; and the capture ofother proteins and the formation of adducts by collagen, also noted, istheorized to occur by a cross-linking reaction, and is believed toaccount for the observed accumulation of albumin and antibodies inkidney basement membrane.

In U.S. Pat. No. 4,758,583, a method and associated agents weredisclosed that served to inhibit the formation of advanced glycosylationendproducts by reacting with an early glycosylation product that resultsfrom the original reaction between the target protein and glucose.Accordingly, inhibition was postulated to take place as the reactionbetween the inhibitor and the early glycosylation product appeared tointerrupt the subsequent reaction of the glycosylated protein withadditional protein material to form the cross-linked late-stage product.One of the agents identified as an inhibitor was aminoguanidine, and theresults of further testing have borne out its efficacy in this regard.

While the success that has been achieved with aminoguanidine and similarcompounds is promising, a need continues to exist to identify anddevelop additional inhibitors that broaden the availability and perhapsthe scope of this potential activity and its diagnostic and therapeuticutility. A further need exists to find agents which not only inhibitthis reaction and its consequences, but agents capable of breaking thecrosslinks formed as a result of pr e-existing advanced glycosylationendproducts, thereby reversing the resultant effects thereof.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method and compositions aredisclosed for the inhibition of formation of advanced glycosylation ofproteins (protein aging) and for breaking the cross-links that formbetween advanced glycosylation (glycation) endproducts (AGEs) or betweenAGEs and other proteins. Advanced glycosylation (glycation) endproductsand cross-linking caused by other reactive sugars present in vivo or infoodstuffs, including ribose, galactose and fructose would also beprevented and reversed by the methods and compositions of the presentinvention.

In particular, the compositions comprise agents for inhibiting theformation of and reversing the pre-formed advanced glycosylation(glycation) endproducts and breaking the subsequent cross-links. Whilenot wishing to be bound by any theory, it is believed that the breakingof the pre-formed advanced glycosylation (glycation) endproducts andcross-links is a result of the cleavage of a dicarbonyl-based proteincrosslinks present in the advanced glycosylation endproducts. Themethods and compositions of this invention are thus directed to agentswhich, by their ability to effect such cleavage, can be utilized tobreak the pre-formed advanced glycosylation endproduct and cross-link,and the resultant deleterious effects thereof, both in vitro and invivo.

Certain of the agents useful in the present invention are members of theclass of compounds known as thiazoliums.

The agents comprise thiazolium compounds having the following structuralformula: ##STR1## wherein R¹ and R² are independently selected from thegroup consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl,lower alkyl, lower alkenyl, or R¹ and R² together with their ringcarbons may be an aromatic fused ring, optionally substituted by one ormore amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula ##STR2## wherein R is a lower alkyl,alkoxy, hydroxy, amino or an aryl group, said aryl group optionallysubstituted by one or more lower alkyl, lower alkoxy, halo,dialkylamino, hydroxy, nitro or alkylenedioxy groups;

a group of the formula

    --CH.sub.2 R'

wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl group;

or a group of the formula ##STR3## wherein R" is hydrogen and R'" is alower alkyl group, optionally substituted by an aryl group, or an arylgroup, said aryl group optionally substituted by one or more loweralkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both loweralkyl groups;

X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion;

and mixtures thereof, and a carrier therefor.

The compounds, and their compositions, utilized in this invention appearto react with an early glycosylation product thereby preventing the samefrom later forming the advanced glycosylation end products which lead tocross-links, and thereby, to molecular or protein aging and otheradverse molecular consequences. Additionally, they react with alreadyformed advanced glycosylation end products to reduce the amount of suchproducts.

The present invention also relates to a method for inhibiting proteinaging and other adverse molecular consequences by contacting theinitially glycosylated molecules at the stage of the early glycosylationproduct with a quantity of one or more of the agents of the presentinvention, or a composition containing the same, and to a method forbreaking the already formed advanced glycosylation end products toreduce the amount of such products by cleaving the c-dicarbonyl-basedcrosslinks present in the advanced glycosylation endproducts. In theinstance where the present method has industrial application, one ormore of the agents may be applied to the proteins in question, forinstance, either by introduction into a mixture of the same in theinstance of a protein extract, or by application or introduction intofoodstuffs susceptible to advanced glycation and crosslinking, all toprevent premature aging and spoilage of the particular foodstuffs, andto reverse the effects of already formed advanced glycosylation endproducts.

The ability to inhibit the formation of advanced glycosylationendproducts, and to reverse the already formed advanced glycosylationproducts in the body carries with it significant implications in allapplications where advanced glycation and concomitant molecularcrosslinking is a serious detriment. Thus, in the area of foodtechnology, for instance, the retardation of food spoilage would conferan obvious economic and social benefit by making certain foods ofmarginal stability less perishable and therefore more available forconsumers. Spoilage would be reduced as would the expense of inspection,removal, and replacement, and the extended availability of the foodscould aid in stabilizing their price in the marketplace. Similarly, inother industrial applications where the perishability of proteins is aproblem, the admixture of the agents of the present invention incompositions containing such proteins would facilitate the extendeduseful life of the same. Presently used food preservatives anddiscoloration preventatives such as sulfur dioxide, known to causetoxicity including allergy and asthma in animals, can be replaced withcompounds such as those described herein.

The present method has particular therapeutic application as theMaillard process acutely affects several of the significant proteinmasses in the body, among them collagen, elastin, lens proteins, and thekidney glomerular basement membranes. These proteins deteriorate bothwith age (hence the application of the term "protein aging") and as aconsequence of diabetes. Accordingly, the ability to either retard orsubstantially inhibit the formation of advanced glycosylationendproducts, and to reduce the amount of cross-links formed betweenadvanced glycosylation endproducts and other proteins in the bodycarries the promise for treatment of the complications of diabetes andaging for instance, and thereby improving the quality and, perhaps,duration of animal and human life.

The present agents are also useful in the area of personal appearanceand hygiene, as they prevent, and reverse, the staining of teeth bycationic anti-microbial agents with anti-plaque properties, such aschlorhexidine.

The invention additionally comprises a novel analytic method for thedetermination of the "breaking" or reversal of the formation ofnon-enzymatic endproducts. In this connection, the invention furtherextends to the identification and use of a novel cross-link structurewhich is believed to represent a significant number of the molecularcrosslinks that form in vitro and in vivo as a consequence of advancedglycation. More particularly, the cross-link structure includes asugar-derived α-dicarbonyl segment or moiety, such as a diketone, thatis capable of cleavage by a dinucleophilic, thiazolium-like compound.Specifically, the cross-link structure may be according to the formula:##STR4## where A and B independently, are sites of attachment to thenucleophilic atom of a biomolecule.

Accordingly, it is-a principal object of the present invention toprovide a method for inhibiting the formation of advanced glycosylationendproducts and extensive cross-linking of molecules, and a method ofbreaking the cross-links formed from pre-existing advanced glycosylationendproducts, that occur as a consequence of the reaction of susceptiblemolecules such as proteins with glucose and other reactive sugars, bycorrespondingly inhibiting the formation of advanced glycosylationendproducts, and breaking the advanced glycosylation mediatedcross-linking that has previously occurred.

It is a further object of the present invention to provide a method asaforesaid which is characterized by a reaction with an initiallyglycosylated protein identified as an early glycosylation product.

It is a further object of the present invention to provide a method asaforesaid which prevents the rearrangement and cross-linking of the saidearly glycosylation products to form the said advanced glycosylationendproducts.

It is a yet further object of the present invention to provide agentscapable of participating in the reaction with the said earlyglycosylation products in the method as aforesaid.

It is a yet further object of the present invention to provide agentswhich break or reverse the advanced glycosylation endproducts formed asa consequence of the aforesaid advanced glycosylation reaction sequenceby cleaving the α-dicarbonyl-based protein crosslinks present in theadvanced glycosylation endproducts.

It is a still further object of the present invention to providetherapeutic methods of treating the adverse consequences of molecular orprotein aging by resort to the aforesaid method and agents.

It is a still further object of the present invention to provide amethod of inhibiting, and reversing, the discoloration of teeth byresort to the aforesaid method and agents.

It is a still further object of the present invention to providecompositions, including pharmaceutical compositions, all incorporatingthe agents of the present invention.

It is still further object of the present invention to provide novelcompounds, as well as processes for their preparation, for use in themethods and compositions of the present invention.

It is a still further object of the present invention to provide novelassays which can be utilized to detect compounds having the ability to"break" or reverse the formation of non-enzymatic glycosylationendproducts and their subsequent cross-links.

It is a yet further object of the present invention to provide across-link structure that is capable of cleavage by the agents thatbreak or reverse the formation of advanced glycosylation endproducts asset forth herein, and the antibodies specific to said cross-linkstructure, and the diagnostic and therapeutic uses thereof.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing description which proceedswith reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is an SDS-PAGE gel showing the CNBrr Peptide Maps of Tail TendonCollagen from Normal and diabetic rates following incubation with3-(2-phenyl-2-oxoethyl)thiazolium bromide (designated as ALT-766) of thepresent invention.

FIG. 2 is an SDS-PAGE gel showing the physical evidence for the breakingof cross-linked AGE-BSA by 3-(2-phenyl-2-oxoethyl)thiazolium bromide, acompound of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, agents, compositions includingpharmaceutical compositions containing said agents and associatedmethods have been developed which are believed to inhibit the formationof advanced glycosylation endproducts in a number of target molecules,including particularly proteins, existing in both animals and plantmaterial, and to reverse the already formed advanced glycosylationendproducts. In particular, the invention relates to a composition whichmay contain one or more agents comprising compounds having the abilityto effect cleavage of α-dicarbonyl-based molecular crosslinks present inthe advanced glycosylation endproducts. Useful agents, for instance,comprise compounds having the structural formula: ##STR5## wherein R¹and R² are independently selected from the group consisting of hydrogen,hydroxy(lower)alkyl, lower aceloxy(lower)alkyl, lower alkyl, loweralkenyl, or R¹ and R² together with their ring carbons may be anaromatic fused ring, optionally substituted by one or more amino, haloor alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula ##STR6## wherein R is a lower alkyl,alkoxy, hydroxy, amino or an aryl group, said aryl group optionallysubstituted by one or more lower alkyl, lower alkoxy, halo,dialkylamino, hydroxy, nitro or alkylenedioxy groups;

a group of the formula

    --CH.sub.2 R'

wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl group;

or a group of the formula ##STR7## wherein R" is hydrogen and R'" is alower alkyl group, optionally substituted by an aryl group, or an arylgroup, said aryl group optionally substituted by one or more loweralkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both loweralkyl groups;

X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion;

and mixtures thereof, and a carrier therefor.

The lower alkyl groups referred to above contain 1-6 carbon atoms andinclude methyl, ethyl, propyl, butyl, pentyl, hexyl, and thecorresponding branched-chain isomers thereof. The lower alkynyl groupscontain from 2 to 6 carbon atoms. Similarly, the lower alkoxy groupscontain from 1 to 6 carbon atoms, and include methoxy, ethoxy, propoxy,butoxy, pentoxy, and hexoxy, and the corresponding branched-chainisomers thereof. These groups are optionally substituted by one or morehalo, hydroxy, amino-or lower alkylamino groups.

The lower acyloxy(lower)alkyl groups encompassed by the above formulainclude those wherein the acyloxy portion contain from 2 to 6 carbonatoms and the lower alkyl portion contains from 1 to 6 carbon atoms.Typical acyloxy portions are those such as acetoxy or ethanoyloxy,propanoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy, and thecorresponding branched chain isomers thereof. Typical lower alkylportions are as described hereinabove.

The aryl groups encompassed by the above formula are those containing6-10 carbon atoms, such as naphthyl, phenyl and lower alkylsubstituted-phenyl, e.g., tolyl and xylyl, and are optionallysubstituted by 1-2 halo, hydroxy, lower alkoxy or di(lower)alkylaminogroups. Preferred aryl groups are phenyl, methoxyphenyl and4-bromophenyl groups.

The halo atoms in the above formula may be fluoro, chloro, bromo oriodo.

For the purposes of this invention, the compounds of formula (I) areformed as biologically and pharmaceutically acceptable salts. Usefulsalt forms are the halides, particularly the bromide and chloride,tosylate, methanesulfonate, and mesitylenesulfonate salts. Other relatedsalts can be formed using similarly non-toxic, and biologically andpharmaceutically acceptable anions.

Of the compounds encompassed by Formula I, certain substituents arepreferred. For instance, the compounds wherein R₁ or R₂ are lower alkylgroups are preferred. Also highly preferred are the compounds wherein Yis an amino group, a 2-amino-2-oxoethyl group, a 2-phenyl-2-oxoethyl ora 2-[substituted phenyl]-2-oxoethyl group.

Representative compounds of the present invention are:

3-aminothiazolium mesitylenesulfonate;

3-amino-4,5-dimethylaminothiazolium mesitylenesulfonate;

2,3-diaminothiazolinium mesitylenesulfonate;

3-(2-methoxy-2-oxoethyl)-thiazolium bromide;

3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium bromide;

3-(2-methoxy-2-oxoethyl)-4-methylthiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium bromide;

3-amino-4-methylthiazolium mesitylenesulfonate;

3-(2-methoxy-2-oxoethyl)-5-methylthiazolium bromide;

(3-(2-phenyl-2-oxoethyl)-5-methylthiazolium bromide;

3-[2-(4'-bromophenyl)-2-oxoethyl]thiazolium bromide;

3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methylthiazolium bromide;

3-[2-(4'-bromophenyl)-2-oxoethyl]-5-methylthiazolium bromide;

3-[2-(4'bromophenyl)-2-oxoethyl]-4,5-dimethylthiazolium bromide;

3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium bromide;

3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)thiazoliumbromide;

3,4-dimethyl-5-(2-hydroxyethyl)thiazolium iodide;

3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide;

3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride;

3-(2-methoxy-2-oxoethyl)benzothiazolium bromide;

3-(2-phenyl-2-oxoethyl)benzothiazolium bromide;

3-[2-(4'bromophenyl)-2-oxoethyl)benzothiazolium bromide;

3-(carboxymethyl)benzothiazolium bromide;

2,3-(diamino)benzothiazolium mesitylenesulfonate;

3-(2-amino-2-oxoethyl)thiazolium bromide;

3-(2-amino-2-oxoethyl)-4-methylthiazolium bromide;

3-(2-amino-2-oxoethyl)-5-methylthiazolium bromide;

3-(2-amino-2-oxoethyl)-4,5-dimethylthiazolium bromide;

3-(2-amino-2-oxoethyl)benzothiazolium bromide;

3-(2-amino-2-oxoethyl)4-methyl-5-(2-hydroxyethyl)thiazolium bromide;

3-amino-5-(2-hydroxyethyl)-4-methylthiazolium mesitylenesulfonate;

3-(2-methyl-2-oxoethyl)thiazolium chloride;

3-amino-4-methyl-5-(2-acetoxyethyl)thiazolium mesitylenesulfonate;

3-(2-phenyl-2-oxoethyl)thiazolium bromide;

3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)thiazolium bromide;

3-(2-amino-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)thiazolium bromide;

2-amino-3-(2-methoxy-2-oxoethyl)thiazolium bromide;

2-amino-3-(2-methoxy-2-oxoethyl)benzothiazolium bromide;

2-amino-3-(2-amino-2-oxoethyl)thiazolium bromide;

2-amino-3-(2-amino-2-oxoethyl)benzothiazolium bromide;

3-[2-(4'-methoxyphenyl) -2-oxoethyl]-thiazolinium bromide;

3-[2-(2',4'-dimethoxyphenyl)-2-oxoethyl]-thiazolinium bromide;

3-[2-(4'-fluorophenyl)-2-oxoethyl]-thiazolinium bromide;

3-[2-(2',4'-difluorophenyl)-2-oxoethyl]-thiazolinium bromide;

3-[2-(4'-diethylaminophenyl)-2-oxoethyl]-thiazolinium bromide;

3-propargyl-thiazolinium bromide ;

3-propargyl-4-methylthiazolinium bromide;

3-propargyl-5-methylthiazolinium bromide;

3-propargyl-4,5-dimethylthiazolinium bromide;

3-propargyl-4-methyl-5-(2-hydroxyethyl)-thiazolinium bromide;

3-(2-[3'-methoxyphenyl]-2-oxoethyl)-thiazolium bromide;

3-(2-[3'-methoxy phenyl]-2-oxoethyl)-4methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3-(2-[3'-methoxyphenyl]-2-oxoethyl)-benzothiazolium bromide;

2,3-diamino-4-chlorobenzothiazolium mesitylenesulfonate;

2,3-diamino-4-methyl-thiazolium mesitylenesulfonate;

3-amino-4-methyl-5-vinyl-thiazolium mesitylenesulfonate;

2,3-diamino-6-chlorobenzothiazolium mesitylenesulfonate;

2,6-diamino-benzothiazole dihydrochloride;

2,6-diamino-3[2-(4'-methoxyphenyl)-2-oxoethyl] benzothiazolium bromide;

2,6-diamino-3[2-(3'-methoxyphenyl)-2-oxoethyl] benzothiazolium bromide;

2,6-diamino-3[2-(4'-diethylaminophenyl)-2-oxoethyl] benzothiazoliumbromide;

2,6-diamino-3(2-(4'-bromophenyl)-2-oxoethyl] benzothiazolium bromide;

2,6-diamino-3(2-(2-phenyl-2-oxoethyl) benzothiazolium bromide;

2,6-diamino-3[2-(4'-fluorophenyl-2-oxoethyl] benzothiazolium bromide;

3-acetamido-4-methyl-5-thiazolyl-ethyl acetate mesitylenesulfonate;

2,3-diamino-5-methylthiazolium mesitylenesulfonate;

3-[2-(2'-naphthyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide;

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide;

3-[2-(2',6'-dichlorophenethylamino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium-bromide;

3-[2-dibutylamino-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide;

3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide;

3-[2-(2',6'-diisopropylanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide;

3-amino-4-methyl-5-[2-(2',6'-dichlorobenzyloxy)ethyl]-thioazoliummesitylenesulfonate;

3-[2-(4'-carbmethoxy-3'-hydroxyanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide;

2,3-diamino-4,5-dimethylthiazolium mesitylene sulfonate;

2,3-diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene sulfonate;

2,3-diamino-5-(3',4'-trimethylenedioxy phenyl)-thiazolium mesitylenesulfonate;

3[2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide;

3-(2-[1',4-benzodioxan-6-yl]-2-oxoethyl)-thiazolium bromide;

3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-thiazolium bromide;

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-thiazoliumbromide;

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-thiazoliumbromide;

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-5-methyl-thiazoliumbromide;

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4,5-dimethyl-thiazoliumbromide;

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-benzothiazoliumbromide;

3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-thiazolinium bromide;

3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliniumbromide;

3-[2-4'-diethylaminophenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliniumbromide;

3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium bromide;

3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-4-methyl-5-vinyl-thiazoliumbromide;

3-(2-tert-butyl-2-oxoethyl)-thiazolium bromide

3-(2-tert-butyl-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide;

3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium chloride;

3-(2',6'-dichlorobenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazoliumchloride;

3-(2'-nitrobenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3[2-(4'-chlorophenyl)-2-oxoethyl]-thiazolium bromide;

3[2-(4'-chlorophenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide; and

3[2-(4'-methoxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide.

Certain of the compounds represented by Formula I are novel compoundswhich represent a further embodiment of the present invention. Thesecompounds are represented by the formula ##STR8## wherein R¹ and R² areindependently selected from the group consisting of hydrogen,hydroxy(lower)alkyl, lower aceloxy(lower)alkyl, lower alkyl, loweralkenyl, or R¹ and R² together with their ring carbons may be anaromatic fused ring, optionally substituted by one or more amino, haloor alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula ##STR9## wherein R is a lower alkyl,alkoxy, hydroxy, amino or an aryl group, said aryl group optionallysubstituted by one or more lower alkyl, lower alkoxy, halo,dialkylamino, hydroxy, nitro or alkylenedioxy groups;

a group of the formula

    --CH.sub.2 R'

wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl group;

or a group of the formula ##STR10## wherein R" is hydrogen and R'" is alower alkyl group, optionally substituted by an aryl group, or an arylgroup, said aryl group optionally substituted by one or more loweralkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both loweralkyl groups;

with the proviso that at least one of Y and Z is an amino group, and thefurther proviso that when Y is amino and R₂ and Z are both hydrogen,then R₁ is other than a lower alkyl group; and

X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion.

Other novel compounds are those of the formula ##STR11## wherein R¹ andR² are independently selected from the group consisting of hydrogen,hydroxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl, or R¹ andR² together with their ring carbons may be an aromatic fused ring;

Z is hydrogen or an amino group;

Y is an alkynylmethyl group, or a group of the formula ##STR12## whereinR" is hydrogen and R'" is a lower alkyl group, optionally substituted byan aryl group, or an aryl group, said aryl group optionally substitutedby one or more lower alkyl, halo, or alkoxylcarbonyl groups; or RI andR'" are both lower alkyl groups; and

X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion.

The above compounds are capable of inhibiting the formation of advancedglycosylation endproducts on target molecules, including, for instance,proteins, as well as being capable of breaking or reversing alreadyformed advanced glycosylation endproducts on such proteins. Thecross-linking of protein by formation of advanced glycosylationendproducts contributes to the entrapment of other proteins and resultsin the development in vivo of conditions such as reduced elasticity andwrinkling of the skin, certain kidney diseases, atherosclerosis,osteoarthritis and the like. Similarly, plant material that undergoesnonenzymatic browning deteriorates and, in the case of foodstuffs,become spoiled or toughened and, consequently, inedible, unpalatable ornon-nutritious. Thus, the compounds employed in accordance with thisinvention inhibit this late-stage Maillard effect and intervene in thedeleterious changes described above, and reduce the level of theadvanced glycosylation endproducts already present in the proteinmaterial.

The rationale of the present invention is to use agents which block, aswell as reverse, the post-glycosylation step, e.g., the formation offluorescent chromophores and cross-links, the presence of which isassociated with, and leads to adverse sequelae of diabetes and aging. Anideal agent would prevent the formation of such chromophores and ofcross-links between protein strands and trapping of proteins onto otherproteins, such as occurs in arteries and in the kidney, and reverse thelevel of such cross-link formation already present.

The chemical nature of the early glycosylation products with which thecompounds of the present invention are believed to react may vary, andaccordingly the term "early glycosylation product(s)" as used herein isintended to include any and all such variations within its scope. Forexample, early glycosylation products with carbonyl moieties that areinvolved in the formation of advanced glycosylation endproducts, andthat may be blocked by reaction with the compounds of the presentinvention, have been postulated. In one embodiment, it is envisionedthat the early glycosylation product may comprise the reactive carbonylmoieties of Amadori products or their further condensation, dehydrationand/or rearrangement products, which may condense to form advancedglycosylation endproducts. In another scenario, reactive carbonylcompounds, containing one or more carbonyl moieties (such asglycolaldehyde, glyceraldehyde or 3-deoxyglucosone) may form from thecleavage of Amadori or other early glycosylation endproducts, and bysubsequent reactions with an amine or Amadori product, may form carbonylcontaining advanced glycosylation products such asalkylformyl-glycosylpyrroles.

Several investigators have studied the mechanism of advancedglycosylation product formation. In vitro studies by Eble et al.,(1983), "Nonenzymatic Glucosylation and Glucose-dependent Cross-linkingof Protein", J. Biol. Chem., 258:9406-9412, concerned the cross-linkingof glycosylated protein with nonglycosylated protein in the absence ofglucose. Eble et al. sought to elucidate the mechanism of the Maillardreaction and accordingly conducted controlled initial glycosylation ofRNase as a model system, which was then examined under varyingconditions. In one aspect, the glycosylated protein material wasisolated and placed in a glucose-free environment and thereby observedto determine the extent of cross-linking.

Eble et al. thereby observed that cross-linking continued to occur notonly with the glycosylated protein but with non-glycosylated proteins aswell. One of the observations noted by Eble et al. was that the reactionbetween glycosylated protein and the protein material appeared to occurat the location on the amino acid side-chain of the protein.Confirmatory experimentation conducted by Eble et al. in this connectiondemonstrated that free lysine would compete with the lysine on RNase forthe binding of glycosylated protein. Thus, it might be inferred fromthese data that lysine may serve as an inhibitor of advancedglycosylation; however, this conclusion and the underlying observationsleading to it should be taken in the relatively limited context of themodel system prepared and examined by Eble et al. Clearly, Eble et al.does not appreciate, nor is there a suggestion therein, of thediscoveries that underlie the present invention, with respect to theinhibition of advanced glycosylation of proteins both in vitro and invivo.

The experiments of Eble et al. do not suggest the reactive cleavageproduct mechanism or any other mechanism in the in vivo formation ofadvanced glycosylation endproducts in which glucose is always present.In fact, other investigators support this mechanism to explain theformation of advanced glycosylated endproducts in vivo (see for example,Hayase et al, J. Biol. Chem., 263:3758-3764 (1989); Sell and Monnier, J.Biol. Chem., 264:21597-21602 (1989); Oimomi et al., Agric. Biol. Chem.,53(6):1727-1728 (1989); and Diabetes Research and Clinical Practice,6:311-313 (1989). Accordingly, the use of lysine as an inhibitor in theEble et al. model system has no bearing upon the utility of thecompounds of the present invention in the inhibition of advancedglycosylated endproducts formation in the presence of glucose in vivo,and the amelioration of complications of diabetes and aging.

While not wishing to be bound by any particular theory as to themechanism by which the compounds of the instant invention reversealready formed advanced glycosylation endproducts, studies have beenstructured to elucidate a possible mechanism. Earlier studies examiningthe fate of the Amadori product (AP) in vivo have identified one likelyroute that could lead to the formation of covalent, glucose-derivedprotein crosslinks. This pathway proceeds by dehydration of the AP viasuccessive beta-eliminations as shown in the Scheme A below. Thus, lossof the 4-hydroxyl of the AP (1) gives a1,4-dideoxy-1-alkylamino-2,3-hexodiulose (AP-dione) (2). An AP-dionewith the structure of an amino-1,4-dideoxyosone has been isolated bytrapping model APs with the AGE-inhibitor aminoguanidine. Subsequentelimination of the 5-hydroxyl gives a1,4,5-trideoxy-1-alkylamino-2,3-hexulos-4-ene (AP-ene-dione) (3), whichhas been isolated as a triacetyl derivative of its 1,2-enol form.Amadori-diones, particularly the AP-ene-dione, would be expected to behighly reactive toward protein crosslinking reactions by serving astargets for the addition of the amine (Lys, His)-, or sulfhydryl(Cys)-based nucleophiles that exist in proteins, thereby producingstable crosslinks of the form (4). ##STR13##

Note that the linear AP-ene-dione of (3) and the stable cross-link of(4) may cyclize to form either 5- or 6-member lactol rings, althoughonly the 6-member cyclic variant is shown in Scheme A set forth above.

The possibility that a major pathway of glucose-derived crosslinkformation proceeds through an AP-ene-dione intermediate was investigatedby experiments designed to test the occurrence of this pathway in vivoas well as to effect the specific cleavage of the resultantα-dicarbonyl-based protein crosslinks. The thiazolium compounds of theinstant invention are thus believed to act as novel "bidentate"nucleophiles, particularly designed to effect a carbon--carbon breakingreaction between the two carbonyls of the crosslink, as shown in SchemeB below under physiological conditions. This scheme shows the reactionof a prototypic α-dione cleaving agent of the formula I,N-phenacylthiazolium bromide, with an AP-ene-dione derived crosslink.##STR14##

A further experiment to elucidate this reaction involves the reaction ofa compound of the formula I, N-phenacylthiazolium bromide, with1-phenyl-1,2-propanedione to produce the predicted fission product,benzoic acid. The reaction between N-phenacylthiazolium bromide and1-phenyl-1,2-propanedione was rapid and readily proceeded, confirmingthis possible mechanism.

Once early, glucose-derived addition products form on proteins, furtherreactions can ensue to effect a covalent, protein-protein crosslinkingreaction. In this regard, a compound of the formula I,N-phenacylthiazolium bromide, was allowed to react with theAGE-crosslinks that form when AGE-modified BSA (AGE-BSA) is allowed toreact with unmodified, native collagen. This resulted in aconcentration-dependent release of BSA from the pre-formed AGE-mediatedcomplexes. Again, this study confirmed that a significant portion of theAGE-crosslinks that form under experimental conditions consist of anu-diketone or related structure that is susceptible to cleavage by theadvantageous bidentate-type molecules of the compounds of formula Iunder physiological conditions.

To confirm that the same situation occurs in vivo, isolated collagenfrom the tail tendons of rats which had been diabetic for 32 weeks weretreated with a compound of the formula I, N-phenacylthiazolium bromide,prior to cyanogen bromide digestion and gel electrophoresis analysis.The subsequent electrophoresis revealed that the treated collagen wasindistinguishable from untreated, non-diabetic (control) collagen, inmarked contrast to the AGE-modified, highly crosslinked,digestion-resistant collagen that is typically isolated from diabeticanimals.

The present invention likewise relates to methods for inhibiting theformation of advanced glycosylation endproducts, and reversing the levelof already formed advanced glycosylation endproducts, which comprisecontacting the target molecules with a composition of the presentinvention. In the instance where the target proteins are contained infoodstuffs, whether of plant or animal origin, these foodstuffs couldhave applied to them by various conventional means a compositioncontaining the present agents.

In the food industry, sulfites were found years ago to inhibit theMaillard reaction and are commonly used in processed and stored foods.Recently, however, sulfites in food have been implicated in severe andeven fatal reactions in asthmatics. As a consequence, the sulfitetreatment of fresh fruits and vegetables has been banned. The mechanismfor the allergic reaction is not known. Accordingly, the presentcompositions and agents offer a nontoxic alternative to sulfites in thetreatment of foods in this manner.

As is apparent from a discussion of the environment of the presentinvention, the present methods and compositions hold the promise forarresting, and to some extent reversing, the aging of key proteins bothin animals and plants, and concomitantly, conferring both economic andmedical benefits as a result thereof. In the instance of foodstuffs, theadministration of the present composition holds the promise forretarding food spoilage thereby making foodstuffs of increased shelflife and greater availability to consumers. Replacement ofcurrently-used preservatives, such as sulfur dioxide known to causeallergies and asthma in humans, with non-toxic, biocompatible compoundsis a further advantage of the present invention.

The therapeutic implications of the present invention relate to thearrest, and to some extent, the reversal of the aging process which has,as indicated earlier, been identified and exemplified in the aging ofkey proteins by advanced glycosylation and cross-linking. Thus, bodyproteins, and particularly structural body proteins, such as collagen,elastin, lens proteins, nerve proteins, kidney glomerular basementmembranes and other extravascular matrix components would all benefit intheir longevity and operation from the practice of the presentinvention. The present invention thus reduces the incidence ofpathologies involving the entrapment of proteins by cross-linked targetproteins, such as retinopathy, cataracts, diabetic kidney disease,glomerulosclerosis, peripheral vascular disease, arteriosclerosisobliterans, peripheral neuropathy, stroke, hypertension,atherosclerosis, osteoarthritis, periarticular rigidity, loss ofelasticity and wrinkling of skin, stiffening of joints,glomerulonephritis, etc. Likewise, all of these conditions are inevidence and tend to occur at an accelerated rate in patients afflictedwith diabetes mellitus as a consequence of this hyperglycemia. Thus, thepresent therapeutic method is relevant to treatment of these and relatedconditions in patients either of advanced age or those suffering fromone of the mentioned pathologies.

Molecular cross-linking through advanced glycosylation product formationcan decrease solubility of structural proteins such as collagen invessel walls and can also trap serum proteins, such as lipoproteins tothe collagen. Also, this may result in increased permeability of theendothelium and consequently covalent trapping of extravasated plasmaproteins in subendothelial matrix, and reduction in susceptibility ofboth plasma and matrix proteins to physiologic degradation by enzymes.For these reasons, the progressive occlusion of diabetic vessels inducedby chronic hyperglycemia has been hypothesized to result from excessiveformation of sugar-derived and particularly, glucose-derivedcross-links. Such diabetic microvascular changes and microvascularocclusion can be effectively prevented and reversed by chemicalinhibition and reversal of the advanced glycosylation product formationutilizing a composition and the methods of the present invention.

Studies indicate that the development of chronic diabetic damage intarget organs is primarily linked to hyperglycemia so that tightmetabolic control would delay or even prevent end-organ damage. SeeNicholls et al., Lab. Invest., 60, No. 4, p. 486 (1989), which discussesthe effects of islet isografting and aminoguanidine in murine diabeticnephropathy. These studies further evidence that aminoguanidinediminishes aortic wall protein cross-linking in diabetic rats andconfirm earlier studies by Brownlee et al., Science, 232:1629-1632(1986) to this additional target organ of complication of diabetes.Also, an additional study showed the reduction of immunoglobulintrapping in the kidney by aminoguanidine (Brownlee et al., Diabetes,(1): 42A (1986)).

Further evidence in the streptozotocin-diabetic rat model thataminoguanidine administration intervenes in the ht development ofdiabetic nephropathy was presented by Brownlee et al., 1988, supra, withregard to morphologic changes in the kidney which are hallmarks ofdiabetic renal disease. These investigators reported that the increasedglomerular basement membrane thickness, a major structural abnormalitycharacteristic of diabetic renal disease, was prevented withaminoguanidine.

Taken together, these data strongly suggest that inhibition and reversalof the formation of advanced glycosylation endproducts (AGEs), by theteaching of the present invention, may prevent, as well as to someextent reverse late, as well as early, structural lesions due todiabetes, as well as changes during aging caused by the formation ofAGEs.

Diabetes-induced changes in the deformability of red blood cells,leading to more rigid cell membranes, is another manifestation ofcross-linking and aminoguanidine has been shown to prevent it in vivo.In such studies, New Zealand White rabbits, with induced, long-termdiabetes are used to study the effects of a test compound on red bloodcell (RBC) deformability (df). The test compound is administered at arate of 100 mg/kg by oral gavage to diabetic rabbits.

A further consequence of diabetes is the hyperglycemia-induced matrixbone differentiation resulting in decreased bone formation usuallyassociated with chronic diabetes. In animal models, diabetes reducesmatrix-induced bone differentiation by 70%.

In the instance where the compositions of the present invention areutilized for in vivo or therapeutic purposes, it may be noted that thecompounds or agents used therein are biocompatible. Pharmaceuticalcompositions may be prepared with a therapeutically effective quantityof the agents or compounds of the present invention and may include apharmaceutically acceptable carrier, selected from known materialsutilized for this purpose. Such compositions may be prepared in avariety of forms, depending on the method of administration. Also,various pharmaceutically acceptable addition salts of the compounds ofFormula I may be utilized.

A liquid form would be utilized in the instance where administration isby intravenous, intramuscular or intraperitoneal injection. Whenappropriate, solid dosage forms such as tablets, capsules, or liquiddosage formulations such as solutions and suspensions, etc., may beprepared for oral administration. For topical or dermal application tothe skin or eye, a solution, a lotion or ointment may be formulated withthe agent in a suitable vehicle such as water, ethanol, propyleneglycol, perhaps including a carrier to aid in penetration into the skinor eye. For example, a topical preparation could include up to about 10%of the compound of Formula I. Other suitable forms for administration toother body tissues are also contemplated.

In the instance where the present method has therapeutic application,the animal host intended for treatment may have administered to it aquantity of one or more of the agents, in a suitable pharmaceuticalform. Administration may be accomplished by known techniques, such asoral, topical and parenteral techniques such as intradermal,subcutaneous, intravenous or intraperitoneal injection, as well as byother conventional means. Administration of the agents may take placeover an extended period of time at a dosage level of, for example, up toabout 30 mg/kg.

As noted earlier, the invention also extends to a method of inhibitingand reversing the discoloration of teeth resulting from nonenzymaticbrowning in the oral cavity which comprises administration to a subjectin need of such therapy an amount effective to inhibit and reverse theformation of advanced glycosylation endproducts of a compositioncomprising an agent of structural Formula I.

The nonenzymatic browning reaction which occurs in the oral cavityresults in the discoloration of teeth. Presently used anti-plaque agentsaccelerate this nonenzymatic browning reaction and further the stainingof the teeth. Recently, a class of cationic anti-microbial agents withremarkable anti-plaque properties have been formulated in oral rinsesfor regular use to kill bacteria in the mouth. These agents, thecationic antiseptics, include such agents as alexidine, cetyl pyridiniumchloride, chlorhexidine gluconate, hexetidine, and benzalkoniumchloride.

Tooth staining by chlorhexidine and other anti-plaque agents apparentlyresults from the enhancement of the Maillard reaction. Nordbo, J. Dent.Res., 58:1429 (1979) reported that chlorhexidine and benzalkoniumchloride catalyze browning reactions in vitro. Chlorhexidine added tomixtures containing a sugar derivative and a source of amino groupsunderwent increased color formation, attributed to the Maillardreaction. It is also known that use of chlorhexidine results in anincreased dental pellicle. Nordbo proposed that chlorhexidine resultedin tooth staining in two ways: first, by increasing formation ofpellicle which contains more amino groups, and secondly, by catalysis ofthe Maillard reaction leading to colored products.

In accordance with this method, the compounds of Formula I areformulated into compositions adapted for use in the oral cavity.Particularly suitable formulations are oral rinses and toothpastesincorporating the active agent.

In the practice of this invention, conventional formulating techniquesare utilized with nontoxic, pharmaceutically acceptable carrierstypically utilized in the amounts and combinations that are well-knownfor the formulation of such oral rinses and toothpastes.

The agent of Formula I is formulated in compositions in an amounteffective to inhibit and reverse the formation of advanced glycosylationendproducts. This amount will, of course, vary with the particular agentbeing utilized and the particular dosage form, but typically is in therange of 0.01% to 1.0%, by weight, of the particular formulation.

The compounds encompassed by Formula I are conveniently prepared bychemical syntheses well-known in the art. Certain of the compoundsencompassed by Formula I are well-known compounds readily available fromchemical supply houses and/or are preparable by synthetic methodsspecifically published therefor. For instance,3,4-dimethyl-5-(2-hydroxyethyl)thiazolium iodide;3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide;3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride; and3-(carboxymethyl)benzothiazolium bromide are obtainable from AldrichChem. Co.

Compounds described in the chemical and patent literature or directlypreparable by methods described therein and encompassed by Formula I arethose such as 3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide and3-benzyl-5-(2-hydroxyethyl)-4-methyl thiazolium chloride [Potts et al.,J. Org. Chem., 41:187-191 (1976)].

Certain of the compounds of formula (I) are novel compounds, notheretofore known in the art. These compounds are those represented bythe formula Ia ##STR15## wherein R¹ and R² are independently selectedfrom the group consisting of hydrogen, hydroxy(lower)alkyl,acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or R¹ and R² togetherwith their ring carbons may be an aromatic fused ring, optionallysubstituted by one or more amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula ##STR16## wherein R is a lower alkyl,alkoxy, hydroxy, amino or an aryl group, said aryl group optionallysubstituted by one or more lower alkyl, lower alkoxy, halo,dialkylamino, hydroxy, nitro or alkylenedioxy groups;

a group of the formula

    --CH.sub.2 R'

wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl group;

or a group of the formula ##STR17## wherein R" is hydrogen and R'" is alower alkyl group, optionally substituted by an aryl group, or an arylgroup, said aryl group optionally substituted by one or more loweralkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both loweralkyl groups;

with the proviso that at least one of Y and Z is an amino group, and thefurther proviso that when Y is amino and R₂ and Z are both hydrogen,then R₁ is other than a lower alkyl group; and

X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion.

Other novel compounds are those of formula I wherein Y is a loweralkynylmethyl group or a group of the formula ##STR18## wherein R" ishydrogen and R'" is a lower alkyl group, optionally substituted by anaryl group, or an aryl group, said aryl group optionally substituted byone or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R'"are both lower alkyl groups.

The compounds of formula I wherein Y is a group of the formula ##STR19##wherein R is a lower alkyl, alkoxy, hydroxy, amino or aryl group; or agroup of the formula

    --CH.sub.2 R'

wherein R' is hydrogen, or a lower alkyl, lower alkynyl or aryl group;

X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion;

can be prepared according to the methods described in Potts et al., J.Org. Chem., 41:187 (1976); and Potts et al., J. Org. Chem., 42:1648(1977), or as shown in Scheme I below. ##STR20## wherein R¹, R², Z, andR are as hereinabove defined, and X is a halogen atom.

In reaction Scheme I, the appropriate substituted thiazole compound offormula II wherein R¹, R² and Z are as hereinbefore defined, is reactedwith the appropriate halo compound of formula III wherein R and X are ashereinbefore defined, to afford the desired compound of formula Iwherein R¹, R², Z, R and X are as hereinbefore defined.

Typically, this reaction is conducted at reflux temperatures for timesof about 1-3 hours. Typically, a polar solvent such as ethanol isutilized for the conduct of the reaction.

The compounds of formula I wherein Y is an amino group can be preparedaccording to the methods described in Tamura et al., Synthesis, 1(1977), or as shown below in Scheme II. ##STR21## wherein R¹, R² and Zare as defined hereinabove

In the reaction shown in Scheme II, typically conducted in an anhydrouspolar solvent at room temperatures, typical reaction temperatures rangefrom room temperature to reflux, and typical times vary from 1 to about4 hours. This reaction affords the mesitylene sulfonate, which can thenbe optionally converted to other thiazolium salts by typical exchangereactions.

The present invention also involves a novel sandwich enzyme immunoassayused to ascertain the ability of test compounds to "break" or reversealready formed advanced glycosylation endproducts by detecting thebreaking of AGE (Advanced glycosylation endproduct) moieties fromAGE-crosslinked protein. This assay comprises:

a) incubation of AGE-modified bovine serum albumin (AGE-BSA) oncollagen-coated wells of microtiter plates for a period of 2-6 hours ata temperature of 37° C.

b) washing of the wells with PBS-Tween;

c) application of the test compounds to the washed wells of step b;

d) incubation of the test compounds applied to the washed wells for anadditional 12-24 hours at a temperature of about 37° C.; and

e) detection of the AGE-breaking using an antibody raised againstAGE-ribonuclease or cross-link breaking with an antibody against BSA.

The following examples are illustrative of the invention.

EXAMPLE 1 3-(2-Methoxy-2-oxoethyl)-thiazolium Bromide

Thiazole, (850 mg, 10 mmol), methyl bromoacetate (1.52, mmol) andabsolute ethanol (50 ml) were refluxed for 2 hours. On cooling, the saltseparated and was recrystallized from absolute ethanol to give the titlecompound (1.59 g), m.p. 189-190° C. (dec).

EXAMPLE 2 3-Amino-4,5-dimethylthiazolium Mesitylenesulfonate

An ice cold solution of the 4,5-dimethyl thiazole (2,26 g, 20 mmol) indry dichloromethane (15 ml) was treated dropwise with a solution ofo-mesitylenesulfonylhydroxylamine (4.3 g, 20 mmol) in drydichloromethane (15 ml). After stirring for 2 hours at room temperature,anhydrous ether (10 ml) was added. On cooling, colorless needles of thetitle product, 3-amino-4,5-dimethyl-thiazolium mesitylenesulfonate,separated (3.48 g), m.p. 165-168° C.

EXAMPLE 3

Using the procedures described above in Examples 1 and 2, the followingcompounds are prepared.

3-amino-thiazolium mesitylenesulfonate, m.p. 102-104° C.

2,3-diamino-thiazolium mesitylenesulfonate, m.p. 173-175° C. (dec).

3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium bromide, m.p. 184-185°C. (dec).

3-(2-methoxy-2-oxoethyl)-4-methylthiazolium bromide, m.p. 149-151° C.(dec).

3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide, m.p. 218-220° C.(dec).

3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium bromide, m.p. 212-213° C.(dec).

3-amino-4-methyl-thiazolium mesitylene sulfonate, m.p. 143-1440C.

3-(2-methoxy-2-oxoethyl)-5-methyl-thiazolium bromide, m.p. 193-194° C.(dec).

3-(2-phenyl-2-oxoethyl)-5-methyl-thiazolium bromide, m.p. 193-194° C.

3-(2-[4¹ -bromophenyl]-2-oxoethyl)-thiazolium bromide, m.p. 269-270° C.(dec).

3-(2-[4¹ -bromophenyl]-2-oxoethyl)-4-methyl-thiazolium bromide, m.p.248-249° C. (dec).

3-(2-[4¹ -bromophenyl]-2-oxoethyl)-5-methyl-thiazolium bromide, m.p.216-217° C.

3-(2-[4¹ -bromophenyl]-2-oxoethyl)-4,5-dimethylthiazolium bromide, m.p.223-224° C. (dec).

3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)-thiazolium bromide,m.p. 137-138° C.

3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)-thiazolium bromide,m.p. 180-181° C.

3-(2-[4-bromophenyl]-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazoliumbromide, m.p. 251-252° C. (dec).

3,4-dimethyl-5-(2-hydroxyethyl)-thiazolium iodide, m.p. 85-87° C.

3-ethyl-5-(2-hydroxyethyl)-4-methyl thiazolium bromide, m.p. 84-85° C.

3-benzyl-5-(2-hydroxyethyl)-4-methyl thiazolium chloride, m.p. 144-146°C.

3-(2-methoxy-2-oxoethyl)-benzothiazolium bromide, m.p. 144-145° C.(dec).

3-(2-phenyl-2-oxoethyl)-benzothiazolium bromide, m.p. 240-241° C. (dec).

3-(2-[4-bromophenyl)-2-oxoethyl)-benzo-thiazolium bromide, m.p. 261-262°C. (dec).

3-(carboxymethyl)-benzothiazolium bromide m.p. 250° C. (dec).

2,3-diamino-benzothiazolium mesitylenesulfonate, m.p. 212-214° C. (dec).

3-(2-amino-2-oxoethyl)-thiazolium bromide, m.p. 205-206° C.

3-(2-amino-2-oxoethyl)-4-methyl-thiazolium bromide, m.p. 220-222° C.

3-(2-amino-2-oxoethyl)-5-methyl-thiazolium bromide, m.p. 179-180° C.

3-(2-amino-2-oxoethyl)-4,5-dimethyl-thiazolium bromide, m.p. 147-148° C.

3-(2-amino-2-oxoethyl)-benzothiazolium bromide, m.p. 222-223° C.

3-(2-amino-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium bromide,m.p. 182-183° C.

3-amino-5-(2-hydroxyethyl)-4-methyl-thiazolium mesitylenesulfonate, m.p.94-95° C. (dec).

3-(2-methyl-2-oxoethyl)thiazolium chloride, m.p. 178-179° C.

3-amino-4-methyl-5-(2-acetoxyethyl)thiazolium mesitylenesulfonate, m.p.118-120° C.

3-(2-phenyl-2-oxoethyl)thiazolium bromide, m.p. 217-218° C.

3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)thiazolium bromide,m.p. 217-218° C.

3-(2-amino-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)thiazolium bromide,m.p. 233-234° C.

2-amino-3-(2-methoxy-2-oxoethyl)thiazolium bromide, m.p. 191-192° C.

2-amino-3-(2-methoxy-2-oxoethyl)benzothiazolium bromide, m.p. 236-237°C.

2-amino-3-(2-amino-2-oxoethyl)thiazolium bromide, m.p. 209-210° C.

2-amino-3-(2-amino-2-oxoethyl)benzothiazolium bromide, m.p. 234-235° C.

3-[2-(4'-methoxyphenyl)-2-oxoethyl]-thiazolinium bromide, m.p. 248-249°C. (dec.);

3-[2-(2',4'-dimethoxyphenyl)-2-oxoethyl]-thiaxolinium bromide, m.p.214-216° C. (dec.);

3-[2-(4'-fluorophenyl-2-oxoethyl]-thiazolinium bromide, m.p. 209-210° C.(dec.);

3-[2-(2',4'-difluorophenyl)-2-oxoeethyl]-thiazolinium bromide, m.p.226-228° C. (dec.);

3-[2-(4'-diethylaminophenyl)-2-oxoethyl]-thiazolinium bromide, m.p.233-235° C. (dec.);

3-propargyl-thiazolium bromide, m.p. 64-66° C.;

3-Propargyl-4-methyl thiazolium bromide, m.p. 213-215° C.;

3-Propargyl-5-methyl thiazolium bromide, m.p. 127-129° C.;

3-Propargyl-4,5-dimethyl thiazolium bromide, m.p. 198-200° C.;

3-Propargyl-4-methyl-5-(2-hydroxyethyl)-thiazolium bromide, m.p.132-134° C.

3-(2-[3'-methoxyphenyl]-2-oxoethyl)-thiazolium bromide, m.p. 224-225°C.;

3-(2-[3'-methoxy phenyl]-2-oxoethyl)-4methyl-5-(2'-hydroxyethyl)-thiazolium bromide. m.p. 164-165° C.;

3-(2-[3'-methoxyphenyl]-2-oxoethyl)-benzothiazolium bromide, m.p.215-217° C.;

2,3-diamino-4-chlorobenzothiazolium mesitylenesulfonate, m.p. 228-230°C.;

2,3-diamino-4-methyl-thiazolium mesitylene sulfonate, m.p. 204-205° C.;

3-amino-4-methyl-5-vinyl-thiazolium mesitylene sulfonate, m.p. 145-147°C.;

2,3-diamino-6-chlorobenzothiazolium mesitylenesulfonate, m.p. 244-246°C.;

2,6-diamino-benzothiazole dihydrochloride, m.p. 318-320° C. (dec.);

2,6-diamino-3[2-(4'-methoxyphenyl)-2-oxoethyl] benzothiazolium bromide,m.p. 243-245° C. (dec.);

2,6-diamino-3[2-(3'-methoxyphenyl)-2-oxoethyl] benzothiazolium bromide,m.p. 217-218° C. (dec.);

2,6-diamino-3[2-(4'-diethylaminophenyl)-2-oxoethyl] benzothiazoliumbromide, m.p. 223-225° C. (dec.);

2,6-diamino-3(2-(4'-bromophenyl)-2-oxoethyl] benzothiazolium bromide,m.p. 258-259° C. (dec.);

2,6-diamino-3(2-(2-phenyl-2-oxoethyl) benzothiazolium bromide, m.p.208-210° C. (dec.);

2,6-diamino-3[2-(4'-fluorophenyl-2-oxoethyl] benzothiazolium bromide,m.p. 251-252° C. (dec.);

3-acetamido-4-methyl-5-thiazolyl-ethyl acetate mesitylenesulfonate, m.p.syrup material;

2,3-diamino-5-methylthiazolium mesitylenesulfonate, m.p. 149-152° C.;

3-[2-(2'-naphthyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 219-220° C.;

3-[2-(3',5'-Di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 206-207° C.;

3-[2-(2',6'-Dichlorophenethylamino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium-bromide,m.p. 193-195° C.;

3-[2-Dibutylamino-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 78-80° C.;

3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 204-206° C.;

3-[2-(2',6'-Diisopropylanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 166-168° C.;

3-amino-4-methyl-5-[2(2',6'-dichlorobenzyloxy)ethyl]-thioazoliummesitylenesulfonate, m.p. 164-166° C.;

3-[2-(4'-carbmethoxy-3'-hydroxyanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 222-223° C.;

2,3-Diamino-4,5-dimethyl thiazolium mesitylene sulfonate, m.p. 166-168°C.;

2,3-Diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene sulfonate,m.p. 132-134° C.;

2,3-Diamino-5-(3',4'-trimethylenedioxy phenyl) thiazolium mesitylenesulfonate, m.p. 224-226° C.;

3-[2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 196-198° C.;

3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 164-166° C.;

3-(2-[1',4'-benzodioxan-6-yl]-2-oxoethyl) thiazolium bromide, m.p.238-239° C.;

3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-thiazolium bromide,m.p. 246-248° C. (dec.);

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-thiazoliumbromide, m.p.

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-thiazoliumbromide, m.p. 226-228° C. (dec.);

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-5-methyl-thiazoliumbromide, m.p. 210-211° C.;

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4,5-dimethyl-thiazoliumbromide, m.p. 243-244° C. (dec.);

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-benzothiazoliumbromide, m.p. 239-294° C. (dec.);

3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-thiazolinium bromide, m.p. 218-220°C. (dec.);

3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliniumbromide, m.p. 178-180° C. (dec.);

3-[2-4'-diethylaminophenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliniumbromide, m.p. 184-186° C. (dec.);

3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium bromide, m.p.176-177° C.;

3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-4-methyl-5-vinyl-thiazoliumbromide, m.p. 208-209° C.;

3-(2-tert-butyl-2-oxoethyl)-thiazolium bromide, m.p. 211-212° C.;

3-(2-tert-butyl-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 186-187° C.;

3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium chloride,m.p. 135-136° C.;

3-(2',6'-dichlorobenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazoliumchloride, m.p. 192-194° C.;

3-(2'-nitrobenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide, m.p.215-216° C.;

3-[2-(4'-chlorophenyl)-2-oxoethyl]-thiazolium bromide, m.p. 239-241° C.(dec.);

3-[2-(4'-chlorophenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumbromide, m.p. 240-251° C. (dec.); and

3[2-(4'-methoxyphenyl)-2-oxoethyl]-4-methyl-5-(2'hydroxyethyl)-thiazolium bromide, m.p. 229-231° C. (dec.).

                  EXAMPLE 4                                                       ______________________________________                                                        mg/tablet                                                     ______________________________________                                        Compound of Formula I                                                                           50                                                            Starch 50                                                                     Mannitol 75                                                                   Magnesium stearate 2                                                          Stearic acid 5                                                              ______________________________________                                    

The compound, a portion of the starch and the lactose are combined andwet granulated with starch paste. The wet granulation is placed on traysand allowed to dry overnight at a temperature of 45° C. The driedgranulation is comminuted in a comminutor to a particle size ofapproximately 20 mesh. Magnesium stearate, stearic acid and the balanceof the starch are added and the entire mix blended prior to compressionon a suitable tablet press. The tablets are compressed at a weight of232 mg. using a 11/32" punch with a hardness of 4 kg. These tablets willdisintegrate within a half hour according to the method described in USPXVI.

    ______________________________________                                                             mg/g                                                     ______________________________________                                        EXAMPLE 5                                                                       Lotion                                                                        Compound of Formula I 1.0                                                     Ethyl alcohol 400.0                                                           Polyethylene glycol 400 300.0                                                 Hydroxypropyl cellulose 5.0                                                   Propylene glycol to make 1.0 g                                                EXAMPLE 6                                                                     Oral Rinse                                                                    Compound of Formula I: 1.4%                                                   Chlorhexidine gluconate 0.12%                                                 Ethanol 11.6%                                                                 Sodium saccharin 0.15%                                                        FD&C Blue No. 1 0.001%                                                        Peppermint Oil 0.5%                                                           Glycerine 10.0%                                                               Tween 60 0.3%                                                                 Water to 100%                                                                 EXAMPLE 7                                                                     Toothpaste                                                                    Compound of Formula I: 5.5%                                                   Sorbitol, 70% in water 25%                                                    Sodium saccharin 0.15%                                                        Sodium lauryl sulfate 1.75%                                                   Carbapol 934, 6% dispersion in 15%                                            Oil of Spearmint 1.0%                                                         Sodium hydroxide, 50% in water 0.76%                                          Dibasic calcium phosphate dihydrate 45%                                       Water to 100%                                                               ______________________________________                                    

EXAMPLE 8 Cross-Linking Inhibition Assay

The following method was used to evaluate the ability of the compoundsof the present invention to inhibit the cross-linking of glycated bovineserum albumin (AGE-BSA) to the rat tail tendon collagen-coated 96-wellplate.

The AGE-BSA was prepared by incubating BSA at a concentration of 200 mgper ml with 200 mM glucose in 0.4M sodium phosphate buffer, pH 7.4 at37° C. for 12 weeks. The glycated BSA was then extensively dialyzedagainst phosphate buffer solution (PBS) for 48 hours with additional 5times buffer exchanges. The rat tail tendon collagen coated plate wasblocked first with 300 μl of superbloc blocking buffer (Pierce #37515X)for one hour. The blocking solution was removed from the wells bywashing the plate twice with PBS-Tween 20 solution (0.05% Tween 20)using a NUNC-multiprobe or Dynatech ELISA-plate washer. Cross-linking ofAGE-BSA (1 to 10 μg per well depending on the batch of AGE-BSA) to rattail tendon collagen coated plate was performed with and without thetesting compound dissolved in PBS buffer at pH 7.4 at the desiredconcentrations by the addition of 50 μl each of the AGE-BSA diluted inPBS or in the solution of test compound at 37° C. for 4 hours. UnbrownedBSA in PBS buffer with or without testing compound were added to theseparate wells as the blanks. The un-cross-linked AGE-BSA was thenremoved by washing the wells three times with PBS-Tween buffer. Theamount of AGE-BSA cross-linked to the tail tendon collagen-coated platewas then quantitated using a polyclonal antibody raised againstAGE-RNase. After a one-hour incubation period, AGE antibody was removedby washing 4 times with PBS-Tween.

The bound AGE antibody was then detected with the addition ofhorseradish peroxidase-conjugated secondary antibody--goat anti-rabbitimmunoglobulin and incubation for 30 minutes. The substrate of2,2-azino-di(3-ethylbenzthiazoline sulfonic acid) (ABTS chromogen)(Zymed #00-2011) was added. The reaction was allowed for an additional15 minutes and the absorbance was read at 410 nm in a Dynatech platereader.

The % inhibition of each test compound was calculated as follows.

    % inhibition={[Optical density (without compound)-optical density (with compound)]/optical density (without compound)}×100%

The IC₅₀ values or the inhibition at various concentrations by testcompounds is as follows:

    ______________________________________                                                                        Relative                                          Cross-link                                                                    Inhibition                                                                   IC.sub.50 (at                                                                Test Compound (mM) 10 mM)                                                   ______________________________________                                        Inhibition Data                                                                 3-amino-4,5-dimethylaminothiazolium                                                                    2.8                                                  mesitylenesulfonate                                                           2,3-diaminothiazolinium mesitylenesulfonate >.10 27%                          3-(2-methoxy-2-oxoethyl)-thiazolium bromide 0.25                              3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium 0.48                          bromide                                                                       3-(2-methoxy-2-oxoethyl)-4-methylthiazolium  58%                              bromide                                                                       3-(2-phenyl-2-oxoethyl)-4-methylthiazolium 5.6                                bromide                                                                       3-(2-phenyl)-2-oxoethyl)-4,5-dimethylthiazolium  37%                          bromide                                                                       3-amino-4-methylthiazolium mesitylenesulfonate  46%                           3-(2-methoxy-2-oxoethyl)-5-methylthiazolium 3.2                               bromide                                                                       (3-(2-phenyl-2-oxoethyl)-5-methylthiazolium 12.6                              bromide                                                                       3-[2-(4'-bromophenyl)-2-oxoethyl]-4-  37%                                     methylthiazolium bromide                                                      3-[2-(4'bromophenyl)-2-oxoethyl]-4,5- 2.92                                    dimethylthiazolium bromide                                                    3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-  38%                                  hydroxyethyl)thiazolium bromide                                               3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2- >10 36%                                hydroxyethyl)thiazolium bromide                                               3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methyl-5- 2.95                            (2-hydroxyethyl)thiazolium bromide                                            3-(2-methoxy-2-oxoethyl)benzothiazolium >10 35%                               bromide                                                                       3-(carboxymethyl)benzothiazolium bromide  16%                                 2,3-(diamino)benzothiazolium mesitylene- 0.0749                               sulfonate                                                                     3-(2-amino-2-oxoethyl)thiazolium bromide 0.53                                 3-(2-amino-2-oxoethyl)-4-methylthiazolium 0.7                                 bromide                                                                       3-(2-amino-2-oxoethyl)-5-methylthiazolium 0.0289                              bromide                                                                       3-(2-amino-2-oxoethyl)-4,5-dimethylthiazolium 9.9                             bromide                                                                       3-(2-amino-2-oxoethyl)benzothiazolium bromide 0.02                            3-(2-amino-2-oxoethyl)4-methyl-5-(2- 1.42                                     hydroxyethyl)thiazolium bromide                                               3-amino-5-(2-hydroxyethyl)-4-methylthiazolium 3.6 × 10.sup.-5                                              mesitylenesulfonate                        3-(2-phenyl-2-oxoethyl)thiazolium bromide 11.1 34%                            3-(2-[3'-methoxyphenyl-2-oxoethyl)-  29%                                      thiazolium bromide                                                            2,3-diamino-4-chlorobenzothiazolium  33%                                      mesitylenesulfonate                                                           2,3-diamino-4-methyl-thiazolium mesitylene  40%                               sulfonate                                                                     3-amino-4-methyl-5-vinyl-thiazolium mesitylene 11.3                           sulfonate                                                                     2,3-diamino-6-chlorobenzothiazolium  23.2                                     mesitylenesulfonate  (2 mm)                                                   2,6-diamino-3[2-(4'-methoxyphenyl)-2-oxoethyl]                                benzothiazolium bromide                                                       2,6-diamino-3(2-(4'-bromophenyl)-2-oxoethyl]                                  benzothiazolium bromide                                                       2,6-diamino-3 [2-(4'-fluorophenyl-2-oxoethyl]                                 benzothiazolium bromide                                                       2,3-diamino-5-methylthiazolium mesitylene-                                    sulfonate                                                                     3-(2-(2'-naphthyl)-2-oxoethyl]-4-methyl-5-(2'-  61%                           hydroxyethyl)-thiazolium bromide                                              3-[2-Dibutylamino-2-oxoethyl]-4-methyl-5-(2'-  0.8%                           hydroxyethyl)-thiazolium bromide  (10 mm)                                     3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-  8.8%                        5-(2'-hydroxyethyl)-thiazolium bromide  (1 mm)                                3-[2-(2',6'-Diisopropylanilino)-2-oxoethyl]-4-  19%                           methyl-5-(2'-hydroxyethyl)-thiazolium bromide                                 3-amino-4-methyl-5-[2(2',6'-dichlorobenzyl-  26.5%                            oxy)ethyl]-thioazolium mesitylenesulfonate  (3 mm)                            3-[2-(4'-carbmethoxy-3'-hydroxyanilino)-2- 1.76                               oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-                                       thiazolium bromide                                                            2,3-Diamino-4,5-dimethyl thiazolium mesitylene  39%                           sulfonate                                                                     2,3-Diamino-4-methyl-5-hydroxyethyl-thiazolium  18%                           mesitylene sulfonate                                                          2,3-Diamino-5-(3',4'-trimethylenedioxy phenyl)-  40% @                        thiazolium mesitylene sulfonate  3 mM                                         3-[2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-  13%                             methyl-5-(2'-hydroxyethyl)-thiazolium bromide                                 3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-  4.4                        4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide                               3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-  45%                        thiazolium bromide                                                            3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-  24% @                         oxoethyl]-4-methyl-thiazolium bromide  0.3 mM                                 3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- 0.78 69% @                     oxoethyl]-5-methyl-thiazolium bromide  1 mM                                   3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- 0.16                           oxoethyl]-4,5-dimethyl-thiazolium bromide                                     3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-thiazolinium  ND                         bromide                                                                       3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-4-methyl-5- 1.53 52% @                   (2'-hydroxyethyl)-thiazolinium bromide  3 mM                                  3-[2-4'-diethylaminophenyl)-2-oxoethyl]-4- 2.8                                methyl-5-(2'-hydroxyethyl)-thiazolinium bromide                               3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl- ND                                  thiazolium bromide                                                            3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxo- ND                            ethyl)-4-methyl-5-vinyl-thiazolium bromide                                  ______________________________________                                    

The above experiments suggest that this type of drug therapy may havebenefit in reducing the pathology associated with the advancedglycosylation of proteins and the formation of crosslinks betweenproteins and other macromolecules. Drug therapy may be used to preventthe increased trapping and crosslinking of proteins that occurs indiabetes and aging which leads to sequelae such as retinal damage, andextravascularly, damage to tendons, ligaments and other joints. Thistherapy might retard atherosclerosis and connective tissue changes thatoccur with diabetes and aging. Both topical, oral, and parenteral routesof administration to provide therapy locally and systemically arecontemplated.

EXAMPLE 9 Cross-Link Breaking Assay

In order to ascertain the ability of the compounds of the instantinvention to "break" or reverse already formed advanced glycosylationendproducts, a novel sandwich enzyme immunoassay was developed whichdetects breaking of AGE (Advanced glycosylation endproduct) moietiesfrom AGE-crosslinked protein. The assay utilizes collagen-coated 96 wellmicrotiter plates that are obtained commercially. AGE-modified protein(AGE-BSA), prepared, for instance, as in Example 8, above, is incubatedon the collagen-coated wells for four hours, is washed off the wellswith PBS-Tween and solutions of the test compounds are added. Followingan incubation period of 16 hours (37° C.) cross-link-breaking isdetected using an antibody raised against AGE-ribonuclease or with anantibody against BSA. Positive results in this assay indicate compoundsthat are capable of reducing the amount of AGE-BSA previouslycrosslinked to the collagen by breaking the crosslinks and allowing theliberated material to be flushed away in subsequent washing steps.Details of the assay are as follows:

Materials

Immunochemicals and Chemicals

Bovine Serum Albumin (Type V), (BSA) Calbiochem

Dextrose

Superbloc, Pierce, Inc.

Rabbit anti-Bovine Serum Albumin

Horseradish Peroxidase (HRP)-Goat-anti-rabbit), Zymed

HRP substrate buffer, Zymed

ABTS chromogen, Zymed

Phosphate Buffer Saline

Tween 20, Sigma

Equipment

ELISA Plate Washer, Dynatech

ELISA Plate Reader, Dynatech

Precision Water Bath

Corning digital pH meter

Glassware and Plasticware

Finneppette Multichannel Pipettor, Baxter

Eppendorf pipettes, Baxter

Eppendorf repeater pipette, Baxter

Pipetter tips for Finneppetter, Baxter

Pipetter tips for Eppendorf, Baxter

Glass test tubes, 13×100 mm; Baxter

Mylar Sealing Tape for 96 well plates, Corning

Biocoat Cellware Rat Tail Collagen Type-1 coated 96-well plates,Collaborative-Biomedical Products

Methods

Preparation of solutions and buffers

1. AGE-BSA stock solutions were prepared as follows. Sodium phosphatebuffer (0.4 M) was prepared by dissolving 6 grams of monobasic sodiumphosphate in 100 ml of distilled water, 7 rams of dibasic sodiumphosphate (0.4 M) in 100 ml of distilled water and adjusting the pH ofthe dibasic solution to 7.4 with the monobasic solution. Sodium azide(0.02 grams) was added per 100 ml volume to inhibit bacterial growth.The BSA solution was prepared as follows: 400 mg of Type V BSA (bovineserum albumin) was added for each ml of sodium phosphate buffer (above).A 400 mM glucose solution was prepared by dissolving 7.2 grams ofdextrose in 100 ml of sodium phosphate buffer (above). The BSA andglucose solutions were mixed 1:1 and incubated at 37° C. for 12 weeks.The pH of the incubation mixture was monitored weekly and adjusted to pH7.4 if necessary. After 12 weeks, the AGE-BSA solution was dialyzedagainst PBS for 48 hours with four buffer changes, each at a 1:500 ratioof solution to dialysis buffer. Protein concentration was determined bythe micro-Lowry method. The AGE-BSA stock solution was aliquoted andstored at -20° C. Dilute solutions of AGE-BSA were unstable when storedat -20° C.

2. Working solutions for crosslinking and breaking studies were preparedas follows. Test compounds were dissolved in PBS and the pH was adjustedto pH 7.4 if necessary. AGE-BSA stock solution was diluted in PBS tomeasure maximum crosslinking and in the inhibitor solution for testinginhibitory activity of compounds. The concentration of AGE-BSA necessaryto achieve the optimum sensitivity was determined by initial titrationof each lot of AGE-BSA.

3. Wash buffer ("PBS-Tween") was prepared as follows. PBS was preparedby dissolving the following salts in one liter of distilled water: NaCl,8 grams; KCl, 0.2 gram, KH₂ PO₄. 1.15 grams; NaN₃, 0.2 gram. Tween-20was added to a final concentration of 0.05% (vol/vol).

4. Substrates for detection of secondary antibody binding were preparedby diluting the HRP substrate buffer 1:10 in distilled water and mixingwith AETS chromogen 1:50 just prior to use.

Assay procedures

1. Biocoat plates were blocked with 300 μl of "Superbloc". Plates wereblocked for one hour at room temperature and were washed with PBS-Tweenthree times with the Dynatech platewasher before addition of testreagents.

2. Each experiment was set up in the following manner. The first threewells of the Biocoat plate were used for the reagent blank. Fiftymicroliters of solutions AGE-BSA were added to test wells in triplicateand only PBS in blank wells. The plate was incubated at 37° C. for fourhours and washed with PBS-Tween three times. Fifty microliters of PBSwas added to the control wells and 50 μl of the test "AGE Cross-linkbreaker" compound was added to the test wells and blank. The plate wasincubated overnight (approximately 16 hours) with the test "AGECross-link breaker" compound, followed by washing in PBS before additionof primary antibody (below).

3. Each lot of primary antibody, either anti-BSA or anti-RNase, wastested for optimum binding capacity in this assay by preparing serialdilutions (1:500 to 1:2000) and plating 50 μl of each dilution in thewells of Biocoat plates. Optimum primary antibody was determined fromsaturation kinetics. Fifty microliters of primary antibody ofappropriate dilution, determined by initial titration, was added andincubated for one hour at room temperature. The plate was then washedwith PBS-Tween.

4. Plates were incubated with the secondary antibody,HRP-(Goat-anti-rabbit), which was diluted 1:4000 in PBS and used as thefinal secondary antibody. The incubation was performed at roomtemperature for thirty minutes.

5. Detection of maximum crosslinking and breaking of AGE crosslinkingwas performed as follows. HRP substrate (100ul) was added to each wellof the plate and was incubated at 37° C. for fifteen minutes. Readingswere taken in the Dynatech ELISA-plate reader. The sample filter was setto "1" and the reference filter was set to "5".

Standard Operating Procedure

Preliminary Steps

1. Titrate each new lot of AGE-BSA preparation as described in Table 4and determine the optimum AGE-BSA concentration for the ELISA assay fromsaturation kinetics.

2. At the beginning of the day, flush the plate washer head with hotwater, rinse with distilled water and 50% ethanol. Fill the bufferreservoir of the plate washer with PBS-Tween (0.05%) and purge thesystem three times before use.

3. Prepare an assay template for setting up the experiment as describedunder "Assay Setup", #2, below.

Assay Setup

1. Warm Superbloc reagent to 37° C. Add 300 μl of Superbloc to each wellof the Biocoat plate and let stand for sixty minutes at 37° C. Wash thewells three times with PBS-Tween (0.05%). Turn the plate 180 degrees andrepeat this wash cycle.

2. Dilute the AGE-BSA in PBS so that 50 μl of the diluted sample willcontain the amount of AGE-BSA necessary for minimum crosslinking andinhibition by pimagedine (aminoguanidine), as determined by initialtitration described above. Prepare negative controls by dissolvingnon-browned BSA in PBS at the same concentration as the AGE-BSA. Add 50μl of AGE-BSA or BSA to each well which correspond to the "AGE-BSA" and"BSA" labels on the template.

3. Dissolve the test compounds in PBS at 30 mM concentration forpreliminary evaluation. The pH must be checked and adjusted to 7.4 whennecessary. Pretreat the collagen-coated plates with AGE-BSA to obtainmaximum crosslinking. Prepare negative controls for inhibitionexperiments by dissolving BSA in the inhibition solution at the sameprotein concentration as that used for AGE-BSA. Add 50 μl of AGE-BSA orBSA in the inhibitor solutions to the wells which correspond to"ALT#+AGE-BSA and to "ALT# blank", respectively, on the template.Incubate the plate at 37° C. for four hours. Following covalent bindingof AGE-BSA to the plates, wash the plates with PBS-Tween in preparationof the detection reaction (below).

4. Binding of primary antibody to the Biocoat plates is carried out asfollows. At the end of the four hour incubation, the wells are washedwith PBS-Tween. Appropriate dilutions (as determined by initialtitration) of the rabbit-anti-AGE-RNase or rabbit-anti-BSA antibodieswere prepared in PBS, and 50 μl is added to each well and the plate isallowed to stand at room temperature for sixty minutes.

5. Secondary antibody binding wells are washed with PBS-Tween and 50microliters HRP (Horseradish Periodase) (Goat anti-rabbit serum) dilutedto 1-4000 in PBS and is added to each well. The plate is allowed tostand at room temperature for 30 minutes.

6. Color development was carried out as follows. Plates are washed as inStep 4 above. Dilute the HRP-substrate buffer 1:10 in water. Add 200 μlof ABTS solution, mix well and add 100 μl of this reagent to each well.Incubate the plate at 37° C. for 15 minutes. Read the optical density at410 nm with the sample filter set to "1" and the reference filter set to"5" on the Dynatech ELISA plate reader. Calculate the percent inhibitionby the compound as described above. Compounds which are found to reducethe amount of immunoreactivity are considered to be therapeuticallyuseful insofar as they reverse and reduce the levels of advancedglycosylation endproducts.

    __________________________________________________________________________                                        Breaking                                     IC.sub.50 (mM) Anti-AGE/Anti-BSA                                             Test Compound Anti-AGE/Anti-BSA (at mM)                                     __________________________________________________________________________    3-aminothiazolium mesitylenesulfonate                                                                   0.005/3.0 71%/67% (30)                                3-amino-4,5-dimethylaminothiazolium  63%/44% (10)                             mesitylenesulfonate                                                           2,3-diaminothiazolinium mesitylenesulfonate 0.28/0.18 79%/90% (10)                                               3-(2-methoxy-2-oxoethyl)-thiazolium                                          bromide  38%/41% (30)                       3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium  63%/47% (30)                 bromide                                                                       3-(2-methoxy-2-oxoethyl)-4-methylthiazolium bromide  54%/51% (30)                                                3-(2-phenyl-2-oxoethyl)-4-methylthiaz                                        olium bromide 0.23/0.30 68%/66% (30)                                           3-(2-phenyl-2-oxoethyl)-4,5-dimethylt                                        hiazolium bromide  56%/ND (30)                                                 3-amino-4-methylthiazolium mesitylene                                        sulfonate  55%/ND (30)                      3-(2-methoxy-2-oxoethyl)-5-methylthiazolium bromide  72%/27% (30)                                                3-[2-(4-bromophenyl)-2-oxoethyl]thiaz                                        olium bromide  76%/25% (30)                 3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2- 14.3/112.0 67%/13% (30)                hydroxyethyl)thiazolium bromide                                               3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium 0.42/0.55 65%/61% (30)                                            chloride                                   3-(2-methoxy-2-oxoethyl)benzothiazolium bromide 1.20/25.9 66%/37% (30)                                           3-(carboxymethyl)benzothiazolium                                             bromide  63.7%/17.9% (30)                   2,3-(diamino)benzothiazolium mesitylenesulfonate  87%/54% (30)                3-(2-amino-2-oxoethyl)-4-methylthiazolium bromide 4.70/38.6 89%/44%                                             (30)                                        3-(2-amino-2-oxoethyl)-4,5-dimethylthiazolium bromide  61%/16% (30)                                              3-(2-amino-2-oxoethyl)benzothiazolium                                         bromide 0.4/0.52 77%/65%                   3-(2-amino-2-oxoethyl)4-methyl-5-(2- 0.012/0.120 65%/57%                      hydroxyethyl)thiazolium bromide                                               3-amino-5-(2-hydroxyethyl)-4- 0.18/0.50 76%/48%                               methylthiazolium mesitylenesulfonate                                          3-(2-methyl-2-oxoethyl)thiazolium chloride 0.83/0.75 56%/93%                  3-(2-phenyl-2-oxoethyl)thiazolium bromide 0.026/0.014 73%/98%                 3-(2-[3'-methoxyphenyl]-2-oxoethyl)-thiazolium bromide  22%/44% (10)                                             2,3-diamino-4-chlorobenzothiazolium                                          mesitylenesulfonate  21%/26 (10)                                               2,3-diamino-4-methyl-thiazolium                                              mesitylenesulfonate  25%/30% (10)                                              3-amino-4-methyl-5-vinyl-thiazolium                                          mesitylenesulfonate ND/2.0 51%/74%                                            (10)                                        2,3-diamino-6-chlorobenzothiazolium mesitylenesulfonate  25%/51 (10)                                             2,6-diamino-3[2-(4'-methoxyphenyl)-2-                                        oxoethyl]  29%/35% (10)                     benzothiazolium bromide                                                       2,6-diamino-3(2-(4'-bromophenyl)-2-oxoethyl]  27%/44% (10)                    benzothiazolium bromide                                                       2,6-diamino-3[2-(4'-fluorophenyl-2-oxoethyl]  24%/40% (10)                    benzothiazolium bromide                                                       2,3-diamino-5-methylthiazolium mesitylenesulfonate  14%/17% (10)                                                 3-[2-(2'-naphthyl)-2-oxoethyl]-4-meth                                        yl-5-(2'-hydroxy-  52%/61% (10)                                                ethyl)-thiazolium bromide                  3-[2-Dibutylamino-2-oxoethyl]-4-methyl-5-(2'-  25%/38% (10)                   hydroxyethyl)-thiazolium bromide                                              3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-  48%/57% (10)                5-(2'-hydroxyethyl)-thiazolium bromide                                        3-[2-(2',6'-Diisopropylanilino)-2-oxoethyl]-4-  31%/48% (10)                  methyl-5-(2'-hydroxyethyl)-thiazolium bromide                                 3-amino-4-methyl-5-[2(2',6'-dichlorobenzyloxy)ethyl]-  31%/54% (10)                                              thioazolium mesitylenesulfonate                                               3-[2-(4'-carbmethoxy-3'-hydroxyanilin                                        o)-2-oxoethyl]-  24%/18% (10)                                                  4-methyl-5-(2'-hydroxyethyl)-thiazoli                                        um bromide                                  2,3-Diamino-4,5-dimethyl thiazolium mesitylene sulfonate  24%/23% (10)                                           2,3-Diamino-4-methyl-5-hydroxyethyl-t                                        hiazolium  20%/18% (10)                     mesitylene sulfonate                                                          2,3-Diamino-5-(3',4'-trimethylenedioxy phenyl)-  13%/42% (1)                  thiazolium mesitylene sulfonate                                               3-[2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-  11%/21% (3)                     methyl-5-(2'-hydroxyethyl)-thiazolium bromide                                 3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-  34%/0 (10)                 4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide                               3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-  17%/18% (10)                                                  thiazolium bromide                         3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-  14%/2% (0.3)                  oxoethyl]-4-methyl-thiazolium bromide                                         3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- 3/0.74 65%/69% (1)                                                oxoethyl]-5-methyl-thiazolium                                                bromide                                     3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-  48%/49% (10)                  oxoethyl]-4,5-dimethyl-thiazolium bromide                                     3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium ND/0.1 62%/82% (1)                                           bromide                                    3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)- ND/0/60% 32%/50%                                          (0.3)                                       4-methyl-5-vinyl-thiazolium bromide                                           3-(2-tert-butyl-2-oxoethyl)-thiazolium bromide  28%/37% (10)                  3-(2-tert-butyl-2-oxoethyl)-4-methyl-5-(2'-  4%/19% (10)                      hydroxyethyl)-thiazolium bromide                                              3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl)-  14%/25% (10)                                                 thiazolium chloride                        3-(2',6'-dichlorobenzyl)-4-methyl-5-(2'-hydroxyethyl)-  6%/27% (10)                                              thiazolium chloride                        3-(2'-nitrobenzyl)-4-methyl-5-(2'-hydroxyethyl)-  11%/13% (10)                thiazolium bromide                                                          __________________________________________________________________________

EXAMPLE 10

To ascertain the ability of the compounds of the invention to decreasethe amount of IgG crosslinked to circulating red blood cells instreptozotocin-induced diabetic rats, was measured by the followingassay. The test compounds are administered to the test animals eitherorally or intraperitoneally, and the blood samples are collected aretested at various times, e.g. 4, 7 or 19 days, after administration toassess efficacy.

Protocol for RBC-IgG assay

A. Preparation of Red Blood Cells

Blood is collected from the rats in heparinized tubes and spun at 2000×g for 10 minutes, and the plasma carefully removed. Then, about 5 ml ofPBS per ml blood is added, gently mixed, and then spun again. Thesupernatant is then removed by aspiration. The wash is then repeated twomore times. Then, 0.2 to 0.3 ml of packed RBC is withdrawn from thebottom of the tube, using a pipette, and added to the PBS to make a 1 to10 dilution. This dilution is then further diluted 1 to 25 and 1 to 50in PBS.

B. Assay set up.

1. Warm Superbloc to 37° C.

2. Take a plate of Multiscreen-HA, 0.45u. Cellulose estermembrane-sealed 96 well plate (Millipore MAHAS45).

3. Wet the wells with 100 ul of PBS.

4. Add 300 ul of superblock to each well and incubate at 37° C. for onehour.

5. Place the plate on the Millititer Vacuum holder, turn on the vacuumand press the plate down once for tight hold. The liquids in the wellswill be suctioned off. Wash the wells with 300 ul of PBS-Tween 0.05%.

6. Turn off the vacuum and add 100 ul of PBS to each well.

7. Gently vortex the RBC samples and pipette 50 ul to the wells, as perthe protocol sheet. Leave first three wells for reagent blanks. Leaveanother three wells for antibody blank.

8. Suction-off the liquid as above and wash the RBCs twice with PBS.

9. Dilute AP(Rb-anti-rat) (Sigma A-6066), 1 to 25000 in PBS.

10. Add 50 ul to the wells and let stand at room temp. for two hours.

11. Suction-off the liquid as above and wash the RBCs twice with PBS.

12. Add pNPP substrate (1 mg/ml in DEA buffer). 100 ul per well.

13. Let the color develop for two hours at 37° C.

14. Place a 96 well corning micrometer plate in the vacuum chamber.

15. Place the sample plate on the vacuum manifold. Make sure the bottomof the plate is completely dry.

16. Apply vacuum for about 5 minutes. Add 100 ul of PBS to all wells andapply vacuum again for 5 minutes. Gently lift the plate and make surethat no liquid drops are hanging at the bottom of the plate. Ifnecessary apply vacuum for few more minutes. Read OD of the solutioncollected in the Corning plate on Dynatech Plate reader Sample filter 1and Ref. filter 4.

17. Calculate percent breaking: 100* (OD410 control-OD410 treated)/OD410control.

Percent inhibition in animals dosed orally at a rate of 10 mg/kg bodyweight are as listed below:

    ______________________________________                                        3-amino-4-methyl-5-vinyl-thiazolium                                                                 11 ± 1 @ 19 days                                       mesitylenesulfonate                                                           3-[2-(2'-naphthyl)-2-oxoethyl]-4- 40 ± 24 @ 19 days                        methyl-5-(2'-hydroxyethyl)-thiazolium                                         bromide                                                                       3-[2-(3',5'-di-tert-butyl-4'-hydroxy- 65 ± 15 @ 19 days                    phenyl)-2-oxoethyl]-5-methyl-thiazolium                                       bromide                                                                       3-(2-phenyl-2-oxoethyl)-4-methyl-5- 58 ± 21 @ 19 days                      vinyl-thiazolium bromide                                                    ______________________________________                                    

The extensive degree of reversal of crosslinking observed in thesestudies underscores two important conclusions by Applicants. First, alarge percentage of cross-links formed in vivo are susceptible to attackand cleavage by the dinucleophilic, thiazolium-based compounds of thepresent invention, and thus, by inference, that these cross-linkscomprise an α-diketone segment consistent with the model shown inSchemes A and B. Second, the crosslink-breaking agents of the presentinvention can act catalytically, in the sense that. a single,dinucleophilic thiazolium-based molecule of the present invention canattack and cause the cleavage of more than one glycation cross-link.

EXAMPLE 11

This example describes the preparation of CNBr peptide maps of rat laidtendon collagen from normal and diabetic animals following treatmentwith a compound of formula I, i.e., 3-(2-phenyl-2-oxoethyl)thiazoliumbromide (ALT 766). Collagen fibers (5 mg) from streptozotocin diabeticrats and age-matched control animals hydrated in land PBS at 60° C. forone hour, the soluble collagen was removed and the pellets were washedseveral times with PBS then treated with3-(2-phenyl-2-oxoethyl)thiazolium bromide at a concentration of 30 mMfor 16 hours. Following incubation, the pellets were centrifuged,washed, and treated with CNBr (40 mg/ml in formic acid at 30° C. for 48hours. The CNBr digests were lyophilized repeatedly to remove CNBr andacid and then subjected to SDS-PAGE (20% acrylamide) under reducingconditions (Lanes 1, 2 and 9, MWS; lane 3, 4 and 5, tail tendon collagenfrom non-diabetic animals with 3 and 5 treated with3-(2-phenyl-2-oxoethyl)thiazolium bromide, 4 was treated with PBS; lanes6,7 and 8, collagen from diabetic animals with 6 and 8 treated with3-(2-phenyl-2-oxoethyl)thiazolium bromide, 7 was treated with PBS). Thegels which result are as shown in FIG. 1.

EXAMPLE 12

Preparation of AGE-BSA and crosslinked-AGE-BSA:

Prepare the following solutions.

1. Buffer: 0.4 M sodium phosphate pH 7.4.

NaH₂ PO₄ :6 g/100 ml

Na₂ HPO₄ :7 g/100 ml

pH of the monbasic sodium phosphate was adjusted to 7.4 with the dibasic0.02 g sodium axide wa added per 100 ml of the buffer.

2. BSA Solution

BSA: Calbiochem Type V; 400 mg/ml in the buffer 1. Total volume prepared50 g/125 ml. Filtered through a 0.45u filter into a sterile one literCorning flask.

3. Glucose solution. 400 uM

Glucose: 400 mM 9 g/125 ml of buffer. Filtered through a 0.45u filterinto one liter Corning sterile flask.

Reaction setup:

BSA and glucose solutions (100 ml each) were mixed in the one literCorning sterile flask, screw-capped tight and incubated at 56° C.without shaking. The bottle was opened once a week to remove aliquotsfor testing. Reaction was continued for 9 weeks when AGE-BSA polymerformation was observed.

Breaking the polymer:

Pieces of AGE-BSA gel was washed with PBS until no more protein wasleached in the supernatant, blotted dry with paper towels. About 50 mgof the washed gel was incubated either with PBs or 10 mm3-(2-phenyl-2-oxoethyl)thiazolium bromide (ALT 766) overnight at 37° C.The supernatants were analyzed by SDS-PAGE and stained with coommassieblue. The resulting gels are shown in FIG. 2.

EXAMPLE 13

To further study the ability of AGE crosslink-inhibiting and reversingagents of the present invention to prevent the discoloration of proteinon a surface, such as that which occurs on the tooth surface, thefollowing surface browning experiment is performed. As a substitute fora pellicle-covered tooth surface, unexposed and developed photographicpaper is used to provide a fixed protein (gelatin, i.e., collagen)surface on a paper backing. Five millimeter circles are punched andimmersed for one week at 50° C. in a solution of 100 mMglucose-6-phosphate in a 0.5 M phosphate buffer, pH 7.4, containing 3 mMsodium azide. Glucose-6-phosphate is a sugar capable of participating innonenzymatic browning at a more rapid rate than glucose. In addition tothe glucose-6-phosphate, chlorhexidine and/or a compound of Formula Iare included. After incubation, the gelatin/paper disks are rinsed withwater, observed for brown color, and photographed.

Incubation of the disks in glucose-6-phosphate alone shows slight browncolor versus disks soaked in buffer alone. Inclusion of chlorhexidine(in the form of Peridex® at a final concentration of 0.04%chlorhexidine) shows significant browning. Addition of a compound ofFormula I to the chlorhexidine completely inhibits browning of thegelatin, as does inclusion of a compound of Formula I in the absence ofchlorhexidine.

The slight brown color formed by the action of glucose-6-phosphate onthe gelatin surface alone and its prevention by a compound of Formula Idemonstrates the utility of the present invention in preventingnonenzymatic browning of tooth surfaces. The enhanced browning in thepresence of chlorhexidine and its prevention with a compound of FormulaI demonstrates the utility of the present invention in preventing theanti-plaque agent-enhanced nonenzymatic browning which occurs withchlorhexidine.

EXAMPLE 14

As a demonstration of the general utility of compounds of the presentinvention to break undesired crosslinks in medically relevantbiomolecules, Applicants conducted the following experiment with theamyloid peptide of Alzheimer's disease. This 14 kDalton peptidecomprises a main constituent of the large, plaque-like aggregates whichform within the brain parenchyma of Alzheimer's disease patients. Thegradual accumulation of such amyloid plaques, together with otherabnormal features such as perivascular amyloid and neurofibrillarytangles, is thought to account for certain of the neurotoxic and otherpathogenic processes of this dementia, which is invariably fatal andpresently incurable. The Alzheimer's amyloid peptide is known toaccumulate AGE modifications in vivo, and upon exposure tophysiologically relevant concentrations of glucose, in vivo, whichglycation enhances the formation of insoluble aggregates of the peptide,reminiscent of Alzheimer's amyloid plaques.

AGE-β-peptide was prepared by incubating an aliquot of the solubleβ-amyloid peptide, synthetically prepared and corresponding in sequenceto the 9-amyloid peptide found in the plaques typical of Alzheimer'sdisease, in a neutral buffered glucose solution for three months,generally as described above for the preparation of AGE-BSA except thatβ-peptide was substituted for BSA as the glycation substrate.

The AGE-β-peptide, glycated and cross-linked after this prolongedexposure to glucose in vivo, was separated from low molecular weightreactants by size exclusion chromatography (e.g. over a PFD-10 column),and iodinated by standard methods to give ¹²⁵ I-AGE-β-peptide as thedesired radiolabeled reagent useful to test or screen compounds formolecular AGE-breaking activity according to the following procedure.Aliquots of ¹²⁵ I-AGE-β-peptide were incubated with or without addedtest compounds of the present invention, at predetermined concentrations(e.g., k 10 mM Compound 766) for a predetermined tine (e.g. overnight),after which a sample of the incubation mixture was prepared fordenaturing gel electrophoresis (SDS-PAGE) and analyzed to determineapparent molecular weight according to well-known procedures.Autoradiograms exposed on the resulting electrophoresis gels werescanned into a digital radiographic imaging and analysis system whichwas used to record radioactivity as a function of apparent molecularweight (electrophoretic mobility in SDS-containing buffer). Inspectionof the results of this experiment showed that if ¹²⁵ I-AGE-β-peptidewere not exposed to an "AGE-breaker" compound of the present invention,it eluted a high molecular weight (>40 kDalton) band, suggesting thatits glycation was accompanied by aggregation and the formation of stablecovalent cross-links. If, however, ¹²⁵ I-AGE-β-peptide was firstincubated in a solution of an AGE crosslink-bearing agent of the presentinvention, the ¹²⁵ I-AGE-β-peptide was significantly disaggregated asshown by the appearance of low molecular weight (>18 kDalton) iodinatedmaterial in the final radiogram. This experiment suggests not only thatdinucleophilic thiazolium-like agents of the present invention can beused to hydrolyze covalent AGE-mediated crosslinks between proteinstrands, but also that such inhibition and reversal of AGEs can reversethe adverse molecular consequences of AGE accumulation on a proteinrelevant to human disease.

EXAMPLE 15

The cross-link structure and related compounds of the present inventionalso find utility as antigens or haptens, to elicit antibodiesspecifically directed thereto. Such antibodies, likewise of the presentinvention, are useful in turn to identify AAA structures of the presentinvention. By constructing immunoassays employing anti-cross-linkstructure antibodies of the present invention, for instance, the degreeto which proteins are modified by such cross-links can be measured. Asdiscussed above, and depending on the half-life of the protein somodified, immunochemical measurement of the cross-link epitopes on aprotein sample, such as hemoglobin, provides an index of recentAGE-formation. Likewise, immunochemical detection of cross-link epitopeson circulating and/or tissue proteins can be used to monitor the courseof therapy with agents of the present invention, which agents aredirected toward inhibition of, and breaking of advanced glycation.

Cross-link-modified BSA for use as an immunogen can be prepared bycoupling a cross-link structure with bovine serum albumin (BSA) usingany of a number of well-known divalent coupling reagents such as acarbodiimide like EDC. Various other haptens, antigens, and conjugatedimmunogens corresponding to the cross-link structures of the presentinvention, including without limitation those described specificallyherein, can conveniently be prepared, either by isolation fromincubation mixtures or by direct synthetic approaches. Thiscross-structure may then be used as an immunogen to raise a variety ofantibodies which recognize specific epitopes or molecular featuresthereof.

In a preferred embodiment, the cross-link structure itself is considereda hapten, which is correspondingly coupled to any of several preferredcarrier proteins, including for instance keyhole limpet hemocyanin(KLH), thyroglobulin, and most preferred, bovine serum albumin (BSA),using a divalent coupling reagents such as EDC, according to protocolswidely circulated in the art.

The cross-link structure, whether alone or coupled to a carrier protein,may be employed in any well-recognized immunization protocol to generateantibodies and related immunological reagents that are useful in anumber of applications owing to the specificity of the antibodies formolecular features of the cross-link structure.

Following a preferred protocol, any of several animal species may beimmunized to produce polyclonal antisera directed against the cross-linkstructure-protein conjugate, including for instance mice, rats,hamsters, goats, rabbits, and chickens. The first of three of theaforesaid animal species are particularly desired choices for thesubsequent production of hybridomas secreting hapten-specific monoclonalantibodies. a The production of said hybridomas from spleen cells ofimmunized animals may conveniently be accomplished by any of severalprotocols popularly practiced in the art, and which describe conditionssuitable for immortalization of immunized spleen cells by fusion with anappropriate cell line, e.g. a myeloma cell line. Said protocols forproducing hybridomas also provide methods for selecting and cloningimmune splenocyte/myeloma cell hybridomas and for identifying hybridomasclones that stably secrete antibodies directed against the desiredepitope(s). Animal species such as rabbit and goat are more commonlyemployed for the generation of polyclonal antisera, but regardless ofwhether polyclonal antisera or monoclonal antibodies are desiredultimately, the hapten-modified carrier protein typically is initiallyadministered in conjunction with an adjuvant such as Complete Freund'sAdjuvant. Immunizations may be administered by any of several routes,typically intraperitoneal, intramuscular or intradermal; certain routesare preferred in the art according to the species to be immunized andthe type of antibody ultimately to be produced. Subsequently, boosterimmunizations are generally administered in conjunction with an adjuvantsuch as alum or Incomplete Freund's Adjuvant. Booster immunizations areadministered at intervals after the initial immunization; generally onemonth is a suitable interval, with blood samples taken between one andtwo weeks after each booster immunization. Alternatively, a variety ofso-called hyperimmunization schedules, which generally feature boosterimmunizations spaced closer together in time, are sometimes employed inan effort to produce anti-hapten antibodies preferentially overanti-carrier protein antibodies.

The antibody titers in post-boost blood samples can be compared forhapten-specific immune titer in any of several convenient formatsincluding, for instance, Ouchterlony diffusion gels and direct ELISAprotocols. In a typical direct ELISA, a defined antigen is immobilizedonto the assay well surface, typically in a 96-well or microtiter plateformat, followed by a series of incubations separated by rinses of theassay well surface to remove unbound binding partners. By way ofnon-limiting example, the wells of an assay plate may receive a dilute,buffered aqueous solution of the hapten/carrier conjugate, preferablywherein the carrier protein differs from that used to immunize theantibody-producing animal to be tested; e.g. serum from AAA/KLHconjugate-immunized animal might be tested against assays wellsdecorated with immobilized AAA/BSA conjugate. Alternatively, the assaysurface may be decorated by incubation with the hapten alone. Generally,the surface of the assay wells is then exposed to a solution of anirrelevant protein, such as casein, to block unoccupied sites on theplastic surfaces. After rinsing with a neutral buffered solution thattypically contains salts and a detergent to minimize non-specificinteractions, the well is then contacted with one of a serial dilutionof the serum prepared from the blood sample of interest (the primaryantiserum). After rinsing again, the extent of test antibodiesimmobilized onto the assay wells by interaction with the desired haptenor hapten/carrier conjugate can be estimated by incubation with acommercially available enzyme-antibody conjugate, wherein the antibodyportion of this secondary conjugate is directed against the species usedto produce the primary antiserum; e.g. if the primary antiserum wasraised in rabbits, a commercial preparation of anti-rabbit antibodiesraised in goat and conjugated to one of several enzymes, such ashorseradish peroxidase, can be used as the secondary antibody. Followingprocedures specified by the manufacturer, the amount of this secondaryantibody can then be estimated quantitatively by the activity of theassociated conjugate enzyme in a calorimetric assay. Many related ELISAor radioimmunometric protocols, such as competitive ELISAs or sandwichELISAs, all of which are well know in the art, may optionally besubstituted, to identify the desired antisera of high titer; that is,the particular antisera which give a true positive result at highdilution (e.g. greater than 1/1000 and more preferably greater than1/10,000).

Similar immunometric protocols can be used to estimate the titer ofantibodies in culture supernatants from hybridomas prepared from spleencells of immunized animals. In so characterizing antisera or hybridomasupernatants, it is desirable to employ a variety of controlincubations, e.g. with different carrier proteins, related butstructurally distinct haptens or antigens, and omitting various reagentsin the immunometric procedure in order to minimize non-specific signalsin the assay and to identify reliable determinations of antibodyspecificity and titer from false positive and false negative results.The types of control incubations to use in this regard are well known.Also, the same general immunometric protocols subsequently may beemployed with the antisera identified by the above procedures to be ofhigh titer and to be directed against specific structural determinantsin the cross-link structures on biological samples, foodstuffs or othercomestibles, or other amine-bearing substances and biomolecules ofinterest. Such latter applications of the desired anti-aldehyde-modifiedAmadori product antibodies, whether polyclonal or monoclonal, togetherwith instructions and optionally with other useful reagents anddiluents, including, without limitation, a set of molecular standards ofthe cross-link structure, may be provided in kit form for theconvenience of the operator.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. An isolated, sugar-derived, nonenzymaticallyformed cross-link of the following structure: ##STR22## wherein A and Bare each independently sites of attachment to a nucleophilic nitrogen orsulphur atom of a biomolecule, said structure capable of cleavage by athiazolium compound.
 2. The cross-link structure of claim 1 wherein A isthe site attachment of an amino acid group to a biomolecule.
 3. Thecross-link structure of claim 1 wherein A is the site of attachment ofan amino acid group to a biomolecule.
 4. The cross-link structure ofclaim 1 wherein wherein said cleavage takes place catalytically.
 5. Thecross-link structure of claim 1 wherein A and B are the sites ofattachment to the protein amino groups.
 6. The crosslink structure ofclaim 5 wherein said cross-link structure forms an intermolecularcrosslink.
 7. The cross link structure of claim 5 wherein said crosslinkstructure forms an intramolecular crosslink.
 8. An agent for reversingpreformed advanced glycosylation crosslinks, comprising a materialcapable of reacting with the crosslink structure of claim
 1. 9. A methodof reversing pre-formed advanced glycosylation crosslinks, comprisingadministering an effective amount of an agent capable of reacting with asugar-derived, nonenzymatically formed crosslink of the followingstructure: ##STR23## wherein A and R are each independently sites ofattachment to a nucleophilic nitrogen or sulphur atom of a biomoleculeand a carrier therefor.
 10. A pharmaceutical composition foradministration to an animal to reverse pre-formed advanced glycosylationcrosslinks, comprising a pharmaceutically effective amount of the agentof claim 8 and a pharmaceutically acceptable carrier.
 11. A method oftreating the complications of diabetes caused by the accumulation ofadvanced glycosylation endproducts in mammals, comprising administeringan effective amount of the composition of claim
 10. 12. A method fortreating a foodstuff containing protein material so as to inhibit theundesired molecular changes therein caused by the presence of advancedglycosylation and products in a target protein within said foodstuff,said method comprising treating the foodstuff within said foodstuff,said method comprising treating the foodstuff with an effective amountof the composition of claim
 9. 13. A method for treating an animal toreverse pre-formed advanced glycosylation end products in a targetbiomolecule within said animal, said method comprising administering tosaid animal an effective amount of the pharmaceutical composition ofclaim
 10. 14. The method of claim 13, wherein said target biomolecule isa protein.
 15. The method of claim 14, wherein said target biomoleculeis selected from the group consisting of collagen, elastin, lensprotein, vessel wall proteins, nerve proteins and glomerular basementmembrane proteins.
 16. The method of claim 13 wherein said agent has theformula (I): ##STR24## wherein R¹ and R² are independently selected fromthe group consisting of hydrogen, hydroxy(lower)alkyl,acyloxy(lower)alkyl, lower alkyl, lower alkenyl, C₆ -C₁₀aryl(lower)alkyl, wherein aryl is optionally substituted with one to twogroups which are halo, hydroxy, lower alkoxy, alkylenedioxy or di(lower)alkylamino groups, or R¹ and R² together with their ring carbons may bea C₆ -C₁₀ aromatic fused ring, optionally substituted by one or moreamino, halo or alkylenedioxy groups;Z is hydrogen or an amino group; Yisamino, a group of the formula --CH₂ C(═O)Rwherein R is a lower alkyl,alkoxy, hydroxy, amino or a C₆ -C₁₀ aryl group, said aryl groupoptionally substituted by one or more lower alkyl, lower alkoxy, halo,dialkylamino, hydroxy, nitro or alkylenedioxy groups and wherein when Ris aryl, at least one of R₁ and R₂ is other than hydrogen and R₁ and R₂are not part of a fused aromatic ring; a group of the formula --CH₂R'wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or C₆ -C₁₀aryl group, said aryl group optionally substituted with one to twogroups which are halo, hydroxy, lower alkoxy or di(lower)alkylaminogroups; or a group of the formula --CH₂ C(═O)N(R")R'"wherein (a) R" ishydrogen and R'" is a lower alkyl group, optionally substituted by a C₆-C₁₀ aryl group, or a C₆ -C₁₀ aryl group, said aryl groups optionallysubstituted by one or more lower alkyl, halo, or (lower) alkoxylcarbonylgroups; or (b) R" and R'" are both lower alkyl groups; X is apharmaceutically acceptable anion; wherein said alkyl, alkynyl andalkoxy groups can be substituted with one or more halo, hydroxy, aminoor lower alkylamino groups; or mixtures of such compounds.
 17. Themethod of claim 16 wherein said compound is a3-benzyl-5-(2-hydroxyethyl)-4-methyl thiazolium compound);3-amino-5-(2-hydroxyethyl)-4-methyl-thiazolium compound; or a3-(2-methyl-2-oxoethyl)thiazolium compound.
 18. The method of claim 16wherein Y is a 2-(substituted or unsubstituted)phenyl-2-oxoethyl group.19. The method of claim 18 wherein Y is 2-phenyl-2-oxoethyl;2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl;2-[4'-bromophenyl]-2-oxoethyl; 2-methoxy-2-oxoethyl; or2-(4'-diethylaminophenyl)-2-oxoethyl.
 20. The method of claim 18 whereinsaid compound is a 3-(2-phenyl-2-oxoethyl)-4-methylthiazolium compound;3-(2-phenyl-2-oxoethyl)thiazolium compound;3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-5-methyl-thiazoliumcompound;3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)]4-methyl-5-vinyl-thiazoliumcompound; 3-(2-phenyl-2-oxoethyl)4-methyl-5-vinyl-thiazolium compound;3-[2-(4'-diethylaminophenyl)-2-oxoethyl]-thiazolium compound or a3-(2-phenyl-2-oxoethyl)-4.5-dimethylthiazolium compound.
 21. The methodof claim 16 wherein Y is a 2-amino-2-oxoethyl group.
 22. The method ofclaim 21 wherein said compound is a3-(2-amino-2-oxoethyl)-benzothiazolium compound; ora3-(2-amino-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium compound.23. The method of claim 16 wherein Y and Z are both amino groups and R¹and R² are independently selected from the group consisting of hydrogen,hydroxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl, loweralkenyl, or R¹ and R² together with their ring carbons may be anaromatic fused ring, optionally substituted by one or more amino, haloor alkylenedioxy groups.
 24. The method of claim 23 wherein saidcompound is a 2.3-diaminothiazolium compound;2,3diamino-4-chlorobenzothiazolium compound;2,3-diamino-4-methyl-thiazolium compound;2,3-diamino-6-chlorobenzothiazolium compound;2,3-diamino-5-methylthiazolium compound; 2,3-diamino-4,5-dimethylthiazolium compound; 2,3-diamino-4-methyl-5-hydroxyethyl-thiazoliumcompound; or a 2,3diamino-5-(3',4'-trimethylenedioxyphenyl) thiazoliumcompound.
 25. The method of claim 16 wherein R¹, R² and Z are hydrogen;Y is a group of the formula --CH₂ C(═O)R wherein R is a lower alkyl,alkoxy, hydroxy, amino or an aryl group, said aryl group optionallysubstituted by one or more lower alkyl, lower alkoxy, halo,dialkylamino, hydroxy, nitro or alkylenedioxy groups, andX is apharmaceutically acceptable anion.
 26. The method of claim 25 whereinsaid compound is a 3-(2-[4'-bromophenyl]-2-oxoethyl)-thiazoliumcompound; 3-[2-(4'-methoxyphenyl)-2-oxoethyl]-thiazolium compound;3-[2-(2',4'-dimethoxyphenyl)-2-oxoethyl]-thiazolium compound;3-[2-(4'-fluorophenyl-2-oxoethyl]-thiazolium compound;3-[2-(2',4'-difluorophenyl)-2-oxoethyl]-thiazolium compound;3-[2-(4'-diethylaminophenyl)-2-oxoethyl]-thiazolium compound;3-(2-[1',4-benzodioxan-6-yl]-2-oxoethyl)-thiazolium compound;3-[2-(3',4'-methylenedioxyphenyl)-2-oxoethyl]-thiazolium compound;3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-thiazoliumcompound; 3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-thiazolium compound:3[2-(4'-chlorophenyl)-2-oxoethyl]-thiazolium compound; or a3-(2-methyl-2-oxoethyl)thiazolium compound.
 27. The method of claim 16wherein R¹, R² and Z are hydrogen; Y is a group of the formula --CH₂C(═O)N(")R'" wherein R' is hydrogen and R'" is a lower alkyl group,optionally substituted by an aryl group, or an aryl group, said arylgroup optionally substituted by one or more lower alkyl, halo, oralkoxylcarbonyl groups; or R" and R'" are both lower alkyl groups; andXis a pharmaceutically acceptable anion.
 28. The method of claim 16wherein R¹, R² and Z are hydrogen;Y is a group of the formula --CH₂ R'wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl group;and X is a pharmaceutically acceptable anion.
 29. The method of claim 16wherein R¹ and R² are independently selected from the group consistingof hydrogen, hydroxy(lower)alkyl, lower acyloxy(lower)alkyl, loweralkyl, lower alkenyl, or R¹ and R² together with their ring carbons maybe an aromatic fused ring; optionally substituted by one or more amino,halo or alkylenedioxy groups;Z is hydrogen; Y is an alkynylmethyl groupor a group of the formula --CH₂ C(═O)N(R")R'" wherein R is an aryl groupoptionally substituted by one or more lower alkyl, halo, oralkoxylcarbonyl groups, or a lower alkyl group substituted by an arylgroup, said aryl group optionally substituted by one or more loweralkyl, halo, or alkoxylcarbonyl groups; and X is a pharmaceuticallyacceptable anion.
 30. The method of claim 29 wherein said compound is a3-[2-(2,6'-dichlorophenethylamino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium-compound;3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumcompound;3-[2-(2',6'-diisopropylanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliumcompound; or a3-[2-(4'-carbmethoxy-3'-hydroxyanilno)-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)-thiazoliumcompound.
 31. The method of claim 16 wherein R¹ and R² are independentlyselected from the group consisting, of hydrogen, hydroxy(lower)alkyl,lower acyloxy(lower)alkyl, lower alkyl, lower alkenyl, or R¹ and R²together with their ring carbons may be an aromatic fused ring;optionally substituted by one or more amino, halo or alkylenedioxygroups;Z is an amino group; Y is a group of the formula --CH₂ C(═O)Rwherein R is a lower alkyl, alkoxy, hydroxy, amino or an aryl group,said aryl group optionally substituted by one or more lower alkyl, loweralkoxy, halo, dialkylamino, hydroxy, nitro or alkylenedioxy groups; andX is a pharmaceutically acceptable anion.
 32. The method of claim 31wherein said compound is a 2-amino-3-(2-methoxy-2-oxoethyl)thiazoliumcompound; 2-amino-3-2-methoxy-2-oxoethyl)benzothiazolium compound;2-amino-3-2-amino-2-oxoethyl)thiazolium compound; or a2-amino-3-(2-amino-2-oxoethyl)benzothiazolium compound.
 33. The methodof claim 31 wherein Y is a 2-(substituted orunsubstituted)phenyl-2-oxoethyl group.
 34. The method of claim 33wherein said compound is a2,6-diamino-3[2-(4'-methoxyphenyl)-2-oxoethyl] benzothiazolium compound;2,6-diamino-3[2-(3'-methoxyphenyl)-2-oxoethyl] benzothiazolium compound;2,6-diamino-3[2-(4'-diethylaminophenyl)-2-oxoethyl] benzothiazoliumcompound; 2,6-diamino-3(2-(4'-bromophenyl)-2-oxoethyl] benzothiazoliumcompound; 2,6-diamino-3(2-(2-phenyl-2-oxoethyl) benzothiazoliumcompound; or a 2,6-diamino-3[2-(4'-fluorophenyl-2-oxoethyl]benzothiazolium compound.
 35. The method of claim 16 wherein R¹ and R²are independently selected from the group consisting of hydrogen,hydroxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl, loweralkenyl, or R¹ and R² together with their ring carbons may be anaromatic fused ring; optionally substituted by one or more amino, haloor alkylenedioxy groups;Z is an amino group; Y is a group of the formula--CH₂ R' wherein R' is hydrogen, a lower alkyl or aryl group; and X is apharmaceutically acceptable anion.
 36. The method of claim 35 whereinthe compound is a3-amino-4-methyl-5-[2-(2',6'-dichlorobenzyloxy)ethyl]-thioazoliumcompound; 3-amino-4-methyl-5-vinyl-thiazolium compound.
 37. A method oftreating kidney damage in a mammal, the method comprising administeringto a mammal in need thereof an effective amount of the composition ofclaim
 10. 38. A method of treating damage to blood vasculature in amammal, the method comprising administering to a mammal in need thereofan effective amount of the composition of claim
 10. 39. The method ofclaim 11, wherein the agent of the administered composition is athiazolium compound.
 40. The method of claim 37, wherein the agent ofthe administered composition is a thiazolium compound.
 41. The method ofclaim 38, wherein the agent of the administered composition is athiazolium compound.
 42. The method of claim 9 wherein the agentcomprises a thiazolium compound capable of reacting with the crosslinkstructure.
 43. The method of claim 18 where Z is hydrogen.
 44. Themethod of claim 43 wherein R¹ and R² are both methyl groups.
 45. Themethod of 44 wherein said compound is3-(2-phenyl-2-oxoethyl)-4,5-dimethyl-thiazolium salt.